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UNIVERSITY  OF  CALIFORNIA 

«EPARTMENT  OF  CIVIL  ENGINEER!** 

BERKELEY,  CALIFORNIA 


Civil  Engineering  Dept 


UNIVERSITY  OF  CAUFQKNIA 

DEPARTMENT  OF  CIVIL  ENGINEERING 

BERKELEY,  CALIFORNIA 


THE  BACTERIOPHAGE 

ITS  ROLE  IN  IMMUNITY 


ENGLISH  EDITION 


THE  BACTEEIOPHAGE 

Its  Role  In  Immunity 

WITH  FOURTEEN  TEXT  ILLUSTRATIONS 


BY 

F.  D'HERELLE 
it 

Pasteur  Institute 


AUTHORIZED   TRANSLATION 

BY 

GEORGE  H.  SMITH,  PH.D. 

ASSISTANT  PROFESSOR  OF  BACTERIOLOGY  AND  PATHOLOGY 
Yale  University  School  of  Medicine 


PUBLISHED  BY 

WILLIAMS  &  WILKINS  COMPANY 

BALTIMORE,  U.  S.  A. 
1922 


ft  I  8 


Engineering 
Library 


COPYRIGHT  1922 
WILLIAMS  &  WILKINS  COMPANY 

Made  in  United  States  of  America 

All  rights  reserved,  including  that  of  translation 

into  foreign  languages,  including 

the  Scandinavian 


COMPOSED  AND  PRINTED  AT  THE 

WAVERLY  PRESS 

BY  THE  WILLIAMS  &  WILKINS  COMPANT 

BALTIMORE,  MD.,  U.  S.  A. 


PREFACE 

In  this  monograph  I  have  collected  and  coordinated  the  several 
notes  and  communications  published,  not  only  by  myself  but 
by  others  also,  since  1917. 

The  study  of  the  phenomenon  was  undertaken  without  any 
preconceived  ideas  regarding  the  nature  of  the  causal  principle 
involved.  Indeed,  what  could  it  matter  whether  the  active 
force  was  a  diastase,  a  living  germ,  or  some  new  property  of  the 
bacterium?  Whatever  it  might  be,  the  interest  would  remain 
the  same.  It  was  only  after  two  years  spent  in  investigation, 
with  the  completion  of  some  hundreds  of  experiments,  each  one 
more  conclusive  than  the  preceding,  that  I  became  convinced 
that  the  cause  of  serial  transmissible  bacteriolysis  could  be  nothing 
other  than  a  living  organism.  And  it  was  not  until  this  time 
that  my  first  communication  on  the  subject  was  presented.  Some 
three  years  later  the  attention  of  others  became  directed  to  the 
subject  and  they,  for  the  most  part,  in  turn  suggested  hypotheses 
as  to  the  nature  of  the  phenomenon  differing  among  themselves 
and  differing  fundamentally  from  that  which  I  had  announced 
and  which,  in  view  of  all  of  the  facts  and  phenomena  involved, 
is  the  only  one  which  is  tenable.  None  of  these  investigators 
have  considered  all  of  the  factors  and  facts  involved  in  the  reac- 
tion; instead,  each  has  selected  a  particular  group  of  facts,  suffi- 
cient to  support  his  thesis,  and  has  neglected  all  other  experi- 
mental data  such  as  would  render  his  hypothesis  inadmissible. 
It  may  be  added  that  all  of  these  hypotheses  were  considered 
prior  to  my  first  publication,  and  the  solution  of  the  question 
required  many  experiments  indeed.  In  spite  of  the  fact  that  the 
results  of  these  experiments  have  been  published  in  various  memo- 
randa none  of  those  who  have  opposed  my  theory  have  refuted 
them,  or  even  alluded  to  them.  Naturally,  in  this  monograph, 
these  experiments  will  be  presented,  experiments  which  by  them- 
selves refute  the  interpretations  of  bacteriophagous  activity  as  a 
diastatic  action,  whether  the  agent  be  derived  from  the  organism 

5 


6  PREFACE 

which  is  defending  itself  or  from  the  bacterium  which  is  causing 
the  infection,  both  of  which  sources  have  been  suggested  by  dif- 
ferent authors. 

It  may  be  that  the  reader  will  find  sometimes  in  the  course  of 
this  discussion  that  I  have  multiplied  evidence  by  repetition, 
or  that  it  is  necessary  to  make  an  effort  to  follow  certain  of  the 
experiments,  so  that  he  is  lost  in  a  maze,  not  only  of  new  phenom- 
mena  for  which  he  is  as  yet  unprepared,  but  also  in  phenomena 
of  extreme  complexity.  The  difficulties  of  exposition  of  the  sub- 
ject will  readily  be  comprehended  if  we  realize  that  up  to  the 
present  time  Bacteriology  has  been  considered  as  a  "  problem 
of  two  bodies,"  bacterium  and  medium,  whether  the  medium  be 
the  organism  parasitized  or  a  culture  fluid.  And  this  problem 
of  the  two  bodies  has  been  indeed  complex.  But  it  is  of  necessity 
much  less  complicated  than  the  "problem  of  three  bodies"  with 
which  we  must  now  be  concerned,  where  we  must  recognize  the 
interactions  between  the  medium — culture  medium  or  organism 
parasitized, — the  bacterium  parasitizing  this  medium,  and  the 
ultramicrobial  bacteriophage  parasitizing  the  bacterium. 

Pan's,  July  1,  1921. 


PREFACE  TO  THE  ENGLISH  EDITION 

The  present  English  edition  of  the  monograph  which  presents 
the  results  of  my  investigations  on  the  Bacteriophage  is  not  a 
simple  translation  of  the  French  edition,  which,  appearing  in 
October,  1921,  was  one  of  the  series  of  monographs  of  the  Pasteur 
Institute.  The  bibliography  has  been  extended,  and  in  many 
of  the  chapters  new  experimental  evidence  has  been  introduced, 
embodying  work  completed  since  the  publication  of  the  French 
edition.  In  addition,  a  wholly  new  chapter  has  been  prepared 
dealing  with  the  " Nature  of  the  Bacteriophage,"  which  pre- 
sents a  statement  of  and  an  analysis  of  the  diverse  hypotheses 
which  have  been  advanced  in  explanation  of  the  intimate  nature 
of  the  principle.  Indeed,  and  this  is  of  some  significance,  this 
is  the  only  point — purely  theoretical  moreover — upon  which  dis- 
cussion of  the  subject  turns.  As  for  the  experimental  facts 
themselves,  they  have  never  been  questioned,  for  all  investiga- 
tors who  have  worked  with  the  bacteriophage  have  confirmed  the 
experimental  results  almost  in  their  entirety. 

It  is  my  hope  that  this  work  will  be  of  interest  to  the  biologist. 
I  hope  especially  that  it  will  stimulate  American  bacteriologists 
in  greater  and  greater  numbers  to  become  interested  in  investiga- 
tive work  upon  the  subject;  one  which  is  new  and  which  promises 
to  be  fruitful  in  theoretical  and  practical  results.  And  this  is 
my  wish  the  more  since,  although  working  for  many  years  in 
France,  the  signatory  of  these  lines  in  the  quality  of  a  Canadian, 
feels  himself  a  part  of  the  great  American  family. 


March  15,  1922 


TABLE  OF  CONTENTS 
Preface 5 


*     *    *     * 


PART  I.    THE  BACTERIOPHAGE 

Introduction 15 

CHAPTER  I.  BACTERIOLYSIS 

Bacteriolysis 23 

Technic  for  the  isolation  of  the  bacteriophage 24 

Technic  for  enhancing  virulence 29 

Enumeration  of  the  bacteriophagous  ultramicrobes 31 

Multiplication  of  the  ultramicroscopic  bacteriophage 34 

The  bacteriophage :  An  obligatory  parasite 38 

The  effect  of  the  condition  of  the  bacteria 40 

The  influence  of  the  medium 42 

Culture  of  the  bacteriophage  on  solid  media:  Isolated  colonies 45 

Effect  of  the  concentration  of  bacteria  in  the  medium;  inhibitory 

effects  of  the  products  of  lysis 49 

Effect  of  external  physical  conditions 53 

Effect  of  antiseptics  upon  lysis 54 

Soluble  substances  of  the  bacteria 57 

The  ultramicrobial  bacteriophage:  An  endoparasite 58 

Bacteriolysis  under  the  microscope 61 

CHAPTER  II.  THE  BACTERIOPHAGE  AND  THE  BACTERIUM 

Virulence  of  the  bacteriophage 66 

Evaluation  of  the  degree  of  virulence 69 

Resistance  of  the  bacterium 70 

The  origin  of  secondary  cultures 73 

Instability  of  mixed  cultures 76 

The  characters  of  mixed  cultures 78 

The  resistant  bacterium 84 

Microscopic  observations 89 

The  acquisition  of  resistance 91 

Production  of  antilysins  by  bacteria 92 

Multiple  cultures 94 

9 


10  CONTENTS 

CHAPTER  III.    VIRULENCE  OF  THE  BACTERIOPHAGE 

Multiple  virulence 96 

Persistence  of  virulence 97 

Bacterial  species  attacked 103 

Bacillus  dysenteriae  Shiga 104 

Bacillus  dysenteriae  Hiss 105 

Bacillus  dysenteriae  Flexner 105 

Bacillus  dysenteriae  "X" 106 

Bacillus  coli 107 

Bacillus  typhosus 107 

Bacillus  paratyphosus  A 108 

Bacillus  paratyphosus  B 108 

Salmonella  (hog  cholera) 108 

Bacillus  typhi  murium. ".  108 

Bacillus  proteus 109 

Bacillus  gallinarum  (Klein),    B.  gallinarum    (Moore);    paragalli- 

narum 109 

Bacterium  diphtheriae 110 

Staphylococcus 110 

Bacterium  of  barbone 110 

Bacillus  pestis Ill 

Bacillus  of  flacherie Ill 

Bacillus  subtilis Ill 

Vibrio  choLerae Ill 

CHAPTER  IV.    THE  BACTERIOPHAGOUS  ULTRAMICROBE 

Morphology 113 

Vitality 115 

Susceptibility  to  different  agents 116 

Unicity  of  the  bacteriophage 120 

The  lysins  of  the  bacteriophage 123 

Opsonic  power  of  the  lysins 125 

CHAPTER  V.    THE  BACTERIOPHAGOUS  ANTISERUM 

Complexity  of  the  antibodies 130 

Antibodies  to  the  bacteria 132 

Antibodies  to  the  bacterial  toxins 132 

Antibodies  to  the  bacteriophagous  ultramicrobes 134 

Antibodies  to  the  lysins 138 

Incidental  conditions  resulting  from  the  existence  of  the  bacteriophage.  141 

CHAPTER  VI.    THE  NATURE  OF  THE  BACTERIOPHAGE 
The  nature  of  the  bacteriophage 144 


CONTENTS  11 

The  possible  hypotheses 146 

Experimental  proofs  of  the  living  nature  of  the  bacteriophage 149 

Refutation  of  the  hypothesis  of  Kabeshima 153 

Refutation  of  the  hypothesis  of  Bordet  and  Ciuca 154 

Refutation  of  the  hypothesis  of  Bail 158 

Refutation  of  the  hypothesis  of  Salimbeni 159 

Conclusions 159 

PART  II.    THE  ROLE  OF  THE  BACTERIOPHAGE  IN  IMMUNITY 
Introduction 163 

CHAPTER  I.    THE  BACTERIOPHAGE  IN  DISEASE 

Choice  of  diseases  to  study 173 

Bacillary  dysentery 176 

Colon  bacillus  infections 189 

Typhoid  fever  and  the  paratyphoid  fevers 189 

Avian  typhosis 204 

Hemorrhagic  septicemia  of  the  buffalo  (barbone) 217 

Bubonic  plague 224 

Flacherie 227 

Conclusions 228 

CHAPTER   II.    THE   BACTERIOPHAGE  IN  THE  HEALTHY  INDIVIDUAL 

The  bacteriophage  in  healthy  man , 229 

The  bacteriophage  in  the  horse 233 

The  bacteriophage  in  the  fowl 236 

The  bacteriophage  in  diverse  animals 237 

Conclusions 239 

CHAPTER  III.    IMMUNIZATION  BY  MEANS  OF  THE  BACTERIOPHAGE 

Immunization  against  avian  typhosis 242 

Immunization  against  barbone 248 

Immunization  against  dysentery 262 

CHAPTER  IV.    THE  BACTERIOPHAGE  AND  IMMUNITY 
Summary  and  conclusions 272 


BIBLIOGRAPHY.  .  283 


PLATE  1 

MULTIPLICATION  OF  THE  ULTRAMICROBIAL  BACTERIOPHAQE 

A  bacterial  suspension  is  inoculated  with  one-billionth  of  a  cubic  centi- 
meter of  a  filtrate  containing  the  Bacteriophage.  From  time  to  time  one- 
fiftieth  of  a  cubic  centimeter  is  taken  from  this  suspension  and  spread  on 
agar.  After  a  period  of  incubation  of  the  agar  tubes  the  following  results 
are  obtained  (reading  from  left  to  right). 

Tube  1.  Planting  made  immediately  after  the  inoculation  of  the  bac- 
teriophage.  A  normal  bacterial  culture  results. 

Tube  2.  Planting  made  lj  hours  after  the  inoculation.  A  normal  bac- 
terial culture  results. 

Tube  3.  Planting  made  1\  hours  after  the  inoculation.  The  layer  of 
bacterial  growth  shows  three  colonies  of  the  bacteriophage. 

Tube  4>  Planting  made  3f  hours  after  the  inoculation.  Confluent 
colonies  of  the  bacteriophage  are  disseminated  throughout  the  bacterial 
growth. 

Tube  5.  Planting  made  after  5  hours.  There  is  no  evidence  of  bacterial 
growth,  the  number  of  ultramicrobes  being  such  that  all  of  the  bacterial 
elements  were  destroyed. 


12 


PLATE  1 


PART  1 
THE  BACTERIOPHAGE 


INTRODUCTION 


HISTORICAL 

Examination  of  the  scientific  literature  discloses  but  two  com- 
munications bearing  on  the  subject  of  the  bacteriophage. 

In  point  of  priority  the  first  is  that  of  Hankin.1  This  author 
states  that  he  detected  in  the  waters  of  certain  rivers  of  India  a 
very  marked  antiseptic  action,  directed  against  bacteria  in 
general,  but  against  the  cholera  vibrio  more  particularly.  Thus, 
for  instance,  the  water  of  the  Jumna  as  it  leaves  the  town  of  Agra 
contains  more  than  100,000  bacteria  per  cubic  centimeter,  while 
at  a  distance  of  5  kilometers  further  down  the  bacterial  content 
is  but  90  to  100. 

With  reference  to  Vibrio  cholerae  in  particular,  his  laboratory 
experiments  gave  results  as  follows.  Specimen  "A"  represents 
the  filtered  water  (filtered  through  porcelain);  specimen  "B"  is 
the  same  filtered  water  after  boiling;  both  specimens  being  inocu- 
lated with  a  culture  of  V.  cholerae. 


NUMBER  C 

)F  ORGANI 

SMS  AFTEB 

0 

1  hour 

2  hours 

3  hours 

4  hours 

25  hours 

49  hours 

Specimen  A  
Specimen  B  

2,500 
5,000 

1,500 
4  000 

1,000 
6,000 

500 
10  000 

0 

6,000 

0 
10,  000 

0 

36,000 

The  germicidal  action  of  the  water  of  these  streams  could 
always  be  detected  but  was  present  to  varying  degrees.  It  is  to 
this  antiseptic  action  that  Hankin  attributes  the  fact  that  the 
ingestion  of  the  water  can  not  be  incriminated  as  the  origin  of 
cholera.  Moreover,  these  streams  have  never  been  the  vectors 
of  epidemics  since  the  propagation  of  such  outbreaks  is  always 
from  downstream  upward. 

1  L'action  bactericide  des  eaux  de  la  Jumna  et  du  Gauge  sur  le  vibrion  du 
cholera.  Ann.  de  Tlnst.  Pasteur,  1896, 10,  511 . 

15 


16  INTKODUCTION 


m  states  that  the  antiseptic  principle  is  destroyed  by 
boiling  and  he  considers  himself  warranted  in  affirming  that 
it  is  a  volatile  substance.  To  my  mind  there  is  no  doubt  that 
this  antiseptic  action  ought  in  reality  to  be  assigned  to 
the  bacteriophage. 

The  second  publication  is  that  of  Twort2  entitled  "An  Investi- 
gation on  the  Nature  of  the  Ultramicroscopic  Viruses."  In  the 
course  of  his  experiments  upon  the  filtrable  virus  of  vaccinia  this 
author  obtained  on  certain  of  his  agar  slants  inoculated  with  the 
glycerinated  vaccinal  pulp,  a  culture  of  a  micrococcus  of  which 
certain  colonies  presented  a  vitreous  and  transparent  aspect. 
The  micrococcus  had  been  replaced  by  fine  granules.  At  other 
times  he  obtained  a  film  of  growth  showing  spots  composed  of 
the  same  vitreous  material.  These  colonies  slowly  spread  over 
the  entire  culture,  the  micrococcus  everywhere  being  transformed 
into  granules.  When  a  pure  culture  of  the  micrococcus  was 
touched  with  a  platinum  wire  which  had  previously  been  in 
contact  with  the  vitreous  material,  a  spot  of  the  same  nature 
developed  and  extended  gradually  over  the  whole  surface.  The 
action  was  feeble  on  cultures  previously  killed.  The  vitreous 
substance,  when  diluted,  passed  through  a  porcelain  filter,  for  a 
drop  of  the  filtrate  transformed  a  normal  healthy  culture  into 
one  of  the  vitreous  appearance.  The  transformation  process 
began  in  isolated  points  and  rapidly  extended  over  the  surface. 
However,  if  some  portion  of  the  normal  culture  never  came  in 
contact  with  the  filtrate,  the  healthy  growth  regained  the  advantage 
and  extended  over  the  vitreous  stratum  but  without  effecting 
its  destruction.  The  material  of  transparent  and  vitreous  nature 
maintained  its  activity  for  at  least  six  months.  It  resisted  a 
temperature  of  52°C.  but  was  destroyed  at  60°C. 

Twort  obtained  similar  results  with  an  organism  of  the  colon 
group,  isolated  from  the  intestinal  mucosa  of  a  dog  affected  with 
Hundeseuche,  and  with  a  large  bacillus  not  belonging  to  the  colon 
group  isolated  from  the  intestinal  contents  of  an  infant  suffering 
from  diarrhea.  In  both  cases  the  material  transformed  the  nor- 
mal culture  into  matter  of  a  vitreous  transparent  aspect. 

2  An  Investigation  on  the  Nature  of  the  Ultramicroscopic  Viruses.  Lan- 
cet, 1915,  ii,  1241  (December  4). 


INTRODUCTION  17 

This  author  reviews  the  different  hypotheses  which  he  consid- 
ered as  possible  causes  in  effecting  the  transformation  of  the  cul- 
tures. The  substance  of  vitreous  appearance,  he  says,  certainly 
contains  an  enzyme  which  is  destroyed  at  60°C.  On  the  other 
hand,  the  material  is  susceptible  of  continued  cultivation,  since 
it  can  be  indefinitely  transplanted  on  a  culture  of  the  micrococ- 
cus.  May  it  be  a  cultivable  enzyme?  May  it  be  living  proto- 
plasm of  indeterminate  form?  May  it  be  an  ancestral  form  of  the 
micrococcus  which  can  not  be  cultivated  in  this  form  but  which 
incites  the  normal  micrococcus  to  take  this  form  of  regression? 
Or,  may  it  be  an  enzyme  secreted  by  the  micrococcus  itself  which 
produces  thus  its  own  destruction,  with  the  formation  of  a  new 
quantity  of  destructive  enzyme?  May  the  substance  of  vitreous 
appearance  be  composed  of  a  filtrable  virus  which  may  be  itself 
the  virus  of  vaccinia  or  a  filtrable  virus  entirely  without  patho- 
genicity  derived  from  the  air  and  which  enters  the  micrococcus 
cultures  by  passing  through  the  cotton  which  closes  the  tubes? 
Twort  did  not  choose  from  these  several  hypotheses  which  he 
formulated,  although  it  seemed  to  him  most  probable  that  the 
vitreous  substance  was  produced  by  the  organism,  the  micrococ- 
cus, itself.  He  indicated  further,  that  he  had  no  idea  as  to  the 
relation  which  might  exist  between  the  bacillus  or  the  micrococ- 
cus, the  vitreous  material,  and  the  disease. 

The  phenomenon  observed  by  Twort  has  been  thus  emphasized 
because  it  involves  a  serial  activity,  and  because,  on  the  other 
hand,  it  is  hardly  probable  that  the  cause  is  a  bacteriophage. 
Indeed,  it  has  been  proved  that  with  a  true  bacteriophage  active 
against  Staphylococcus  albus  the  phenomenon  described  by  Twort 
can  not  be  reproduced.  The  very  peculiar  characters,  and  indeed, 
the  characteristics  presented  by  the  phenomenon  observed  by 
Twort  render  confusion  of  this  principle  with  the  bacteriophage 
impossible.  The  difference  in  thermal  death  points,  amounting 
to  15°C.,  between  the  two  principles  (the  bacteriophage  becomes 
inactive  only  at  about  75°C.)  is  alone  sufficient  to  differentiate 
them.  According  to  some  experiments  which  I  have  performed 
the  facts  observed  by  Twort  may  be  ascribed  to  a  fragmentation 
of  the  bacteria;  it  is  only  necessary  to  use  the  ultramicroscope  to 
see  that  the  "vitreous  substance' '  is  composed  of  very  minute 
cocci. 


18  INTRODU  CTION 

However  that  may  be,  the  intensity  of  the  bacteriophagous 
action  is  sometimes  of  such  violence  that  it  must  have  been  ob- 
served by  many  bacteriologists  in  the  course  of  their  investiga- 
tions even  though  the  nature  of  the  phenomenon  and  its  mechan- 
ism were  not  understood.  For  example,  I  have  been  informed 
that  in  Haffkine's  laboratory,  it  has  been  noted  several  times  that 
cultures  of  the  plague  bacillus  in  bouillon  underwent  clarification, 
the  medium  becoming  perfectly  limpid  within  the  space  of  a  few 
hours.  Not  knowing  the  reason  for  this  curious  phenomenon  the 
cultures  were  termed  "  suicides."  For  such  reactions  the  bac- 
teriophage  was  certainly  the  cause. 

Another  observation  of  the  same  nature  is  reported  by  Eliava, 
who,  being  in  charge  of  the  examination  of  the  water  of  the  Koura 
river  at  Tiflis,  noted  the  following  phenomenon.  The  suspected 
water  under  examination  was  added  to  a  peptone  solution.  After 
a  few  hours  of  incubation  a  specimen  taken  from  the  surface  of 
the  medium  for  microscopic  examination  showed  very  numerous 
vibrios  of  normal  morphology.  Planted  upon  agar,  this  speci- 
men yielded  upon  incubation  a  growth  of  dull  appearance  which 
microscopically  appeared  to  be  a  culture  of  vibrios.  Twelve 
hours  later,  in  so  far  as  the  peptone  water  was  concerned,  all 
trace  of  the  vibrios  had  disappeared.  This  experiment,  repeatedly 
performed,  always  gave  the  same  result;  it  was  impossible  to 
secure  a  culture  of  the  vibrio.  Although  starting  a  normal  de- 
velopment, later,  within  a  few  hours,  the  vibrio  had  disappeared. 
This  phenomenon  remained  unexplained  until  the  findings  with 
reference  to  the  bacteriophage  were  published. 

In  fact,  it  is  certain  that  a  large  number  of  bacteriologists, 
indeed,  it  may  be  said  all  bacteriologists, — and  reasons  for  this 
statement  will  appear  in  the  course  of  this  discussion, — have 
accidentally  encountered  this  strange  phenomenon.  Seen  at 
times  in  a  fluid  medium,  at  other  times  on  a  solid  medium,  such 
reactions  have  repeatedly  been  observed  but  their  study  has  been 
neglected  since  their  importance  was  not  recognized. 

FUNDAMENTAL  EXPERIMENT 

The  experiment  which  served  as  a  point  of  departure  for  sub- 
sequent work  was  as  follows.  An  adult,  suffering  with  a  severe 


INTRODUCTION  19 

dysentery  (B.  dysenteriae  Shiga)  was  under  treatment  in  the 
Pasteur  Hospital.  Each  day  an  examination  of  the  feces  from 
this  patient  was  made  by  the  inoculation  of  bouillon  with  some 
of  the  fecal  material.  After  incubation  at  37°C.  for  over  night 
the  growth  was  filtered  through  a  Chamberland  filter.  To  a 
second  tube  of  broth,  previously  inoculated  with  a  culture  of  the 
Shiga  bacillus,  twelve  drops  of  the  filtrate  were  added  and  the 
culture  so  treated  was  returned  to  the  incubator.  Throughout 
the  period  of  the  infection,  all  of  the  tubes,  prepared  each  day 
in  the  same  manner,  yielded  normal  growths  of  the  dysentery 
bacillus.  One  day,  examination  of  the  tube  prepared  the  day 
before  showed  no  growth,  and  investigation  showed  that  the 
patient  presented  symptoms  indicative  of  marked  improvement. 
Definite  convalescence  rapidly  followed.  The  bouillon  which 
had  been  inoculated  with  both  culture  and  filtrate  was  to  all 
appearances  sterile,  and  to  this  was  again  added  a  suspension  of 
Shiga  bacilli  taken  from  a  young  agar  culture.  The  inoculation 
was  sufficiently  heavy  to  present  a  definite  turbidity,  but  after 
incubation  for  twelve  hours  it  was  again  clear.  The  excreta 
from  which  the  filtrates  were  prepared  contained,  then,  a  prin- 
ciple which  dissolved  the  dysentery  organisms. 

When  a  drop  of  the  lysed  culture  was  added  to  a  young  bouillon 
culture  of  the  Shiga  bacilli  this  culture  in  turn  became  dissolved. 
In  the  same  way,  several  successive  passages  were  accomplished, 
introducing  each  time  a  drop  of  the  culture  previously  lysed  into 
a  fresh  culture  of  the  Shiga  strain.  Instead  of  losing  in  potency 
through  such  passages  it  increased  in  lytic  capacity;  the  dissolv- 
ing action  being  accomplished  more  and  more  rapidly.  From 
this  it  was  evident  that  the  lytic  principle  derived  from  the  ex- 
creta was  capable  of  cultivation  in  series. 

When  a  very  minute  amount  (0.00,001  cc.)  of  one  of  these 
lysed  cultures  was  added  to  a  young  broth  culture  of  the  Shiga 
bacillus  and  then  this  mixture  was  tested  immediately  and  again 
after  incubation  periods  of  one,  two,  and  three  hours,  a  drop  of 
the  material  being  inoculated  on  to  agar  slants,  the  latter  showed 
after  incubation  very  interesting  characteristics.  In  the  first 
tube  thus  inoculated  the  agar  was  covered  by  a  normal  film  of 
dysentery  bacilli,  but  with  two  circular  areas  about  2  mm.  in 


20  INTRODUCTION 

diameter  entirely  free  of  any  evidence  of  bacterial  growth.  The 
second  tube,  inoculated  with  material  taken  one  hour  after  the 
admixture  of  culture  and  lytic  agent,  presented  six  of  the  clear 
plaques.  In  the  third  tube  there  were  about  100;  and  finally, 
on  the  fourth  there  was  no  apparent  growth. 

Here  was  new  evidence  that  the  lytic  principle  actually  multi- 
plied, and  furthermore,  that  this  principle  actually  existed  in 
particulate  form.  The  element  from  which  the  lytic  phenomenon 
originated  was  composed  of  masses  which  were  deposited  upon 
the  agar  in  definite  points.  Each  mass  was  capable  of  multi- 
plication since,  independent  of  the  action  in  series,  it  yielded  a 
colony.  It  could  be  considered  as  nothing  other  than  a  ferment 
or  a  living  being  parasitic  on  the  bacteria.  But  it  is  impossible 
to  comprehend  a  soluble  ferment — a  diastase — as  multiplying 
in  the  form  of  granulations  and  concentrating  its  activities  in 
limited,  clearly  defined  points. 

As  we  will  see  in  the  course  of  this  work  all  the  experiments, 
without  a  single  exception,  are  in  accord  in  showing  that  the 
principle  acts  as  a  virus;  and,  indeed,  as  a  virus  which  presents 
all  the  chief  characteristics  of  organisms,  including  the  fixation 
of  complement  with  an  antiserum. 

In  employing  the  word  "microbe,"  or  better  "ultramicrobe," 
following  the  happy  expression  of  Calmette,  I  give  to  this  word 
its  true  meaning:  "minute  living  being,"  without  any  suggestion 
as  to  what  kingdom  it  may  belong.  Is  it  a  bacterium,  a  proto- 
zoon,  or  a  yeast?  That  must  be  ignored.  Its  dimensions  are 
too  small  to  permit  the  determination  of  this  question  by  direct 
microscopic  observation.  May  it  be  a  cell,  infinitely  small,  an 
organite,  derived  from  a  superior  organism;  a  cell  indefinitely 
cultivable  in  series  in  vitro  at  the  expense  of  bacteria  and  main- 
taining itself  as  an  autonomous  being?  This  is  hardly  probable, 
but  it  is  never  permissible  to  reject  a  priori  any  conception  which 
accords  with  the  known  facts.  Experiment  has  shown  that  this 
lytic  principle,  which  has  been  termed  Bacteriophagum  intestinale 
or  bacteriophage,  is  a  particle  which  proliferates  at  the  expense 
of  bacteria;  and,  as  a  result,  is  capable  of  assimilation  and  is 
indefinitely  cultivable  in  series  in  vitro  in  the  form  of  a  filtrable 
substance.  It  behaves  like  living  matter  because  assimilation 
and  reproduction  are  fundamental  characteristics  of  life. 


INTRODUCTION  21 

This  introductory  discussion  should  not  be  concluded  without 
a  word  on  the  subject  of  the  term  "Bacteriophage,"  a  term  which 
has  been  criticized.  The  suffix  "phage"  is  not  used  in  its  strict 
sense  of  "to  eat,"  but  in  that  of  "developing  at  the  expense  of;" 
a  sense  that  is  frequently  used  elsewhere  in  scientific  termi- 
nology. Certain  protozoa,  for  example,  are  parasitized  by  the 
Nucleophaga  which  develop  within  the  interior  of  the  nucleus. 
This  is  precisely  the  interpretation  to  be  given  the  term  "phage" 
in  the  word  "Bacteriophage." 


CHAPTER   I 

BACTERIOLYSIS 

Bacteriolysis.  Technic  for  Isolating  the  Bacteriophage.  Enhancement 
of  Virulence.  Technic  for  Enumeration.  Multiplication  at  the 
Expense  of  the  Bacteria  in  a  Fluid  Medium.  The  Bacteriophage;  an 
Obligatory  Parasite.  Effect  of  the  Condition  of  the  Bacterium.  Effect 
of  the  Medium.  Cultivation  on  Solid  Media;  Isolated  Colonies.  Effect 
of  the  Concentration  of  Bacteria  in  the  Medium.  Destructive  Action  of 
the  Secretory  Products  of  the  Bacteriophage.  Effect  of  External 
Physical  Conditions.  Effect  of  Antiseptics.  The  Soluble  Bacterial 
Substance.  The  Bacteriophagous  Ultramicrobe ;  an  Internal  Parasite. 
Bacteriolysis  under  the  Microscope. 

BACTERIOLYSIS 

It  is  expedient  to  define,  at  the  beginning  of  this  work,  what 
is  meant  by  the  word  "bacteriolysis"  for  it  is  a  scientific  term 
used  in  a  somewhat  equivocal  manner. 

The  term  "autolysis"  was  introduced  into  science  byJacoby 
as  a  substitute  for  the  word  "autophagy"  which  had  previously 
been  employed  to  designate  the  process  of  softening;  the  tendency 
toward  a  liquefaction,  more  or  less  marked,  such  as  is  produced 
by  a  yeast  isolated  upon  a  nutrient  medium.  The  term  autophagy, 
which  presumed  nothing  as  to  the  final  condition  of  the  process, 
is  more  suitable  certainly,  than  that  of  autolysis.  Etymologi- 
cally  the  latter  signifies  auto-dissolution,  whereas  as  a  matter  of 
fact,  the  process  of  autolysis,  as  it  occurs  with  bacteria  and  yeasts 
even  if  prolonged  for  several  months,  never  results  in  a  complete 
cellular  dissolution.  The  end  product  is  a  semifluid  mass,  which, 
examined  microscopically,  shows  cellular  debris  along  with  a 
greater  or  less  number  of  cells  more  or  less  profoundly  modified. 
The  degree  of  disintegration  depends  somewhat  upon  the  type: 
of  bacterial  cells  employed.  Autolysis,  then,  is  characterized, 
not  by  an  actual  dissolution,  but  by  a  disintegration,  a  cellular 
fragmentation  with  a  partial  dissolution  of  certain  elements. 
Even  in  the  most  favorable  cases,  when  the  autolysis  is  considered 

23 


24  THE  BACTERIOPHAGE 

complete,  it  is  attended  by  the  formation  of  an  amorphous  mass 
insoluble  in  the  fluid.  If  the  significance  of  the  term  "lysis" 
is  indeed  exact  in  autolysis  where  there  is  a  partial  dissolution, 
it  is  by  no  means  the  same  in  "  bacteriolysis"  as  this  is  understood 
by  many  authors.  Treatises  dealing  with  immunity  speak  of 
"  bacteriolytic  sera,"  realizing  explicitly  that  the  reactions  be- 
tween antigen  and  antibody  never  consist  of  a  digestion.  How 
then,  under  these  conditions,  is  it  possible  to  have  a  dissolution? 
And,  in  fact,  none  is  ever  observed. 

The  action  manifested  by  the  bacteriophage  is  wholly  different. 
It  comprises  phenomena  of  which  the  final  result  is  a  digestion 
such  as  leads  to  a  total  dissolution  of  the  bacterial  bodies.  It 
is  a  bacteriolysis  in  the  true  sense.  At  first  I  considered  desig- 
nating this  new  phenomenon  by  a  new  word,  "  bacteriophagy" 
for  example,  since  the  word  bacteriolysis  is  often  employed  to 
designate  processes  differing  entirely  from  dissolution.  But 
since  the  use  of  too  frequent  neologisms  might  distract  the  reader 
I  have  felt  that  the  abuse  which  has  been  made  of  the  term 
"  bacteriolysis"  may  be  only  a  transitory  one  and  that  the  passage 
of  time  will  soon  cause  to  be  forgotten  the  phenomenon  of  so- 
called  bacteriolysis  without  a  dissolution  of  the  bacteria. 

To  summarize:  the  phenomena  which  we  will  consider  have 
nothing  in  common  with  that  which  is  usually  designated  by  the 
terms  " lysis"  and  "bacteriolysis."  Here,  the  term  "lysis" 
should  always  be  taken  in  its  strict  etymological  sense  of  a  com- 
plete dissolution.  A  bacterial  culture  in  bouillon  or  a  suspension 
of  bacteria  in  a  fluid  where  bacteriolysis,  as  we  understand  it, 
takes  place,  completely  clears,  without  residue.  The  fluid  be- 
comes as  limpid  as  it  was  prior  to  its  inoculation  with  culture. 

TEGHNIC  FOR  THE  ISOLATION  OF  THE  BACTERIOPHAGE 

All  bacteriological  laboratories  possess  the  equipment  required 
for  the  isolation  and  cultivation  of  the  bacteriophage.  For  isola- 
tion a  filtering  apparatus  is  indispensable.  (I  have  employed  the 
model  of  Martin,  with  Chamberland  L2  and  L3  bougies.)  For 
culture  media  a  peptone  bouillon,  or  such  a  bouillon  incorporated 
into  a  2  per  cent  agar,  is  adequate. 


BACTERIOLYSIS  25 

The  active  bacteriophage  may  be  sought  for  in  materials  which 
require  some  preliminary  treatment,  since  in  their  natural  physi- 
cal state  they  may  not  lend  themselves  readily  to  nitration. 
The  following  types  of  material  may  be  examined  as  a  source 
of  the  bacteriophage. 

1.  A  sterile  fluid;  sterile  in  the  sense  in  which  the  word1  is 
usually  employed;  for  example,  blood,  or  an  organic  fluid  col- 
lected aseptically.     With  such  no  treatment  is  necessary. 

2.  The  material  to  be  examined  may  be  a  clear  liquid  but  not 
sterile.     With  this,  filtration  will  eliminate  the  bacteria  while 
the  bacteriophage  passes  through  into  the  filtrate. 

3.  The  material  may  show  a  homogeneous  turbidity;  as  a 
bacterial  culture.     Here,  direct  filtration  results  in  an  early  occlu- 
sion of  the  pores  of  the  bougie.     Thus,  it  is  desirable  to  resort  to 
a  preliminary  filtration.     The  following  method  of  treatment 
is  most  satisfactory. 

Provide  a  funnel  with  a  folded  niter  paper  sufficiently  large  to  receive 
at  one  time  the  entire  volume  to  be  filtered.  Fill  the  filter  with  water  to 
which  has  been  added  a  small  amount  of  infusorial  earth.  When  the  water 
has  passed  through,  the  paper  is  left  coated  with  a  thin  layer  of  the  infuso- 
rial earth,  thus  rendering  the  paper  less  permeable.  Through  this  the 
material  to  be  examined  is  filtered  prior  to  filtration  through  the  bougie. 

4.  The  material  may  be  a  fluid  holding  in  suspension  organic 
particles,  or  it  may  be  matter  more  or  less  solid  in  nature.    This 
is  the  type  of  substance  most  frequently  examined;  such  as  fecal 

1  In  the  course  of  this  work  I  find  myself  frequently  in  difficulty  in  the 
exposition  of  facts  because  of  certain  expressions  which  have  been  appro- 
priated to  describe  certain  conditions.  I  shall  apply  the  word  sterile  to 
a  medium  which  contains  no  visible  microscopic  organisms  or  organisms 
capable  of  cultivation  upon  artificial  media.  An  ultrasterile  medium  is 
one  which  contains  no  ultramicrobes.  A  substance  containing  the  virus  of 
measles,  for  example,  is  sterile  but  not  ultrasterile,  since  it  is  still  capable 
of  transmitting  measles  although  it  contains  nothing  visible  or  cultivable. 
Media  containing  the  bacteriophage  are  likewise  sterile  in  the  bacteriologi- 
cal sense  of  the  word,  since  they  are  perfectly  limpid  and  since  the  germ 
which  they  contain  can  not  be  cultivated  alone  upon  artificial  media  of  any 
kind.  But  such  a  medium  is  not  ultrasterile,  for  it  does  contain  a  principle 
which  will  grow  at  the  expense  of  bacteria,  just  as  the  virus  of  measles  will 
grow  at  the  expense  of  higher  organisms. 


26  THE   BACTERIOPHAGE 

material  more  or  less  fluid,  pasty,  or  solid;  or  excreta  admixed  to 
a  greater  or  less  degree  with  earth,  organic  debris,  etc.  In  such 
a  case  it  is  necessary  to  disintegrate  as  completely  as  possible 
the  material  to  be  examined. 

To  effect  such  a  disintegration  the  most  simple  procedure  consists  in  care- 
fully suspending  the  material  in  bouillon,  about  5  gm.  to  50  cc.  of  the 
medium,  and  incubating  this  suspension  at  37°C .  for  from  twelve  to  eighteen 
hours.  The  bacterial  fermentations  which  ensue,  because  of  the  diverse 
organisms  introduced  into  the  medium,  lead  to  a  sufficient  disintegration. 
Upon  removal  from  the  incubator  the  material  may  be  treated,  as  indicated 
above,  by  nitration  through  infusorial  earth  and  a  bougie. 

If  the  material  under  examination  contains  the  bacteriophage 
and  has  been  subjected  to  filtration,  the  ultramicrobe  will  be 
found  in  the  filtrate.  The  methods  of  purification  outlined  above 
are  applicable  to  two  purposes,  (A)  the  detection  of  a  bacterio- 
phage active  toward  a  given  bacterial  type,  and  (B)  to  test  the 
activity  of  a  bacteriophage,  either  upon  diverse  organisms  or 
against  a  single  bacterial  strain  of  indeterminate  type. 

A.  The  first  case  is  the  more  simple  and  will  be  considered 
first,  taking  as  an  example  the  detection  of  a  bacteriophage  active 
against  B.  dysenteriae  Shiga.  The  day  before  the  test  is  to  be 
made  an  agar  slant  is  inoculated  with  the  dysentery  strain.  From 
this  fresh  culture,  on  the  day  of  the  test,  four  tubes  of  peptone 
broth  are  inoculated.2  To  the  first  of  these  tubes  is  added  one 
drop  of  the  filtrate,  to  the  second,  ten  drops,  and  to  the  third, 
two  cubic  centimeters.  One  tube,  simply  inoculated  with  the  dys- 
entery organism,  serves  as  a  control.  The  tubes  are  incubated 
at  37°C.  After  twelve  to  eighteen  hours  one  of  several  results 
may  be  observed :  the  three  tubes  (in  addition  to  the  control  tube) 
may  all  show  a  turbidity  due  to  the  growth  of  the  dysentery 
organism,  only  one  or  two  of  the  tubes  may  be  turbid,  or  the 
three  tubes  may  be  clear. 

*  As  will  be  shown  later  this  bouillon  should  be  alkaline  in  reaction. 
The  ordinary  neutral  bouillon  (I  have  always  used  by  preference  the 
bouillon  of  Martin)  to  which  is  added  6  cc.  of  N/l  NaOH  per  liter  is  per- 
fectly satisfactory.  This  is,  moreover,  the  degree  of  alkalinity  most 
frequently  employed  in  bacteriological  work. 


BACTERIOLYSIS  27 

1.  All  three  tubes  are  turbid.  From  such  a  result  it  may  not 
be  concluded  that  an  active  bacteriophage  is  not  present,  for  if 
a  complete  lysis  of  the  bacteria  is  taken  as  the  only  criterion  for 
determining  its  presence  the  bacteriophage  will,  in  a  majority  of 
cases,  be  overlooked.  Lysis  is  but  a  single  fact  in  the  midst  of 
a  very  complex  group  of  phenomena.  If  the  three  tubes  are 
turbid  take  about  0.02  cc.  from  each  of  the  tubes  by  means  of  a 
platinum  loop  and  spread  over  the  surfaces  of  three  tubes  of 
slanted  agar.  If,  after  incubation,  these  tubes  present  normal 
cultures  of  the  dysentery  bacillus  the  result  of  the  test  is  negative. 
That  is,  the  original  material  did  not  contain  an  active  bacterio- 
phage for  B.  dysenteriae  Shiga.  The  presence  of  the  bacteriophage 
in  active  form  is  indicated  by  an  abnormal  appearance  of  the 
growth  as  it  develops  on  the  agar.  In  accordance  with  the  num- 
ber of  bacteriophagous  ultramicrobes  present  the  aspect  of  the 
culture  will  vary.  The  layer  of  bacillary  growth  may  show  one, 
or  several,  circular  areas  where  the  surface  of  the  agar  appears 
devoid  of  growth.  Or,  the  culture  may  appear  broken  up,  or 
corroded,  as  a  result  of  the  confluence  of  the  areas.  Indeed, 
there  may  be  only  fragments  of  culture  or  even  isolated  colonies 
remaining.  When  the  number  of  ultramicrobes  is  still  greater 
the  slant  may  be  free  of  any  evidence  of  bacterial  growth. 

As  will  be  shown,  each  strain  of  bacteriophage  is  endowed  with 
an  individual  degree  of  virulence,  the  word  "  virulence"  to  be 
taken  in  its  true  meaning,  that  is,  "ability  to  multiply  at  the 
expense  of  the  parasitized  being."  Certain  races  of  the  bac- 
teriophage multiply  rapidly,  others  increase  but  slowly.  The 
first  possess  a  high  degree  of  virulence  toward  the  bacterium 
provided  for  their  development;  the  second  possess  but  a  feeble 
virulence.  We  will  elsewhere  return  to  this  subject  of  the  viru- 
lence of  the  bacteriophage.  It  is  mentioned  here  simply  to  ex- 
plain the  reason  why  strains  of  the  bacteriophage  show  varia- 
bility in  growth  when  isolation  is  attempted.  The  diameter  of 
the  clear  areas,  varying  with  the  individual  strain  of  bacterio- 
phage from  a  fraction  of  a  millimeter  (the  bacterial  growth  appears 
as  though  sprinkled  with  pin  point  areas)  up  to  4  to  5  mm.,  gives 
a  measure  of  virulence;  the  larger  the  area  the  higher  the  viru- 
lence. We  shall  see  that  whatever  the  virulence  of  a  particular 


28  THE   BACTERIOPHAGE 

strain  of  the  bacteriophage  when  it  comes  from  the  organism  it 
can  be  enhanced  in  vitro. 

2.  The  first  or  first  and  second  tubes  only  give  a  culture  of  the 
dysentery  bacillus.    Here  the  filtrate  contains  a   bacteriophage 
of  average  or  high  activity.     It  is  only  necessary  to  proceed  as 
is  indicated  in  the  following  case  to  secure  areas  of  bacteriophagous 
growth  on  agar. 

3.  All  three  tubes  remain  clear.     This  indicates  the  presence 
of  a  bacteriophage  of  extremely  high  activity.     Confirmation  is 
simple.     It  consists  in  taking  three  tubes  of  bouillon,  in  adding 
to  them  a  suspension  of  young  bacilli  taken  from  an  agar  slant 
in  a  concentration  to  give  a  slight  turbidity.     Introduce  into  each 
of  these  tubes  a  drop  of  the  fluid  from  each  of  the  three  tubes 
which  had  remained  clear,  shake,  and  then  immediately  distribute 
a  loopful  of  each  upon  the  surface  of  an  agar  slant.    Both  sets 
of  tubes  are  incubated.     After  twelve   to   eighteen  hours  the 
three  bouillon  tubes  will  be  limpid;  the  three  agar  slants  will 
present  the   appearance   already   described  for   cultures   of   B. 
dysenteriae  admixed  with  the  bacteriophage. 

B.  dysenteriae  Shiga  has  been  taken  as  an  example,  although 
whatever  may  be  the  bacterial  type  against  which  an  active 
bacteriophage  is  sought  the  technic  for  isolation  remains  essen- 
tially the  same.  The  medium  is  inoculated  with  the  bacterium 
in  question,  as,  for  example,  with  B.  pestis  if  a  bacteriophage  active 
for  the  plague  bacillus  is  sought. 

B.  Instead  of  determining  if  a  given  material  contains  a  bac- 
teriophage active  for  a  certain  bacterium,  it  may  be  desirable 
to  ascertain  if  a  bacteriophage  which  has  been  isolated  possesses 
an  activity  for  several  bacterial  types  at  the  same  time.  In 
this  instance  three  tubes  with  each  of  the  bacterial  types  to  be 
investigated  may  be  prepared.  Thus,  to  investigate  the  activity 
of  an  intestinal  bacteriophage  against  B.  dysenteriae  Shiga,  B. 
dysenteriae  Flexner,  B.  dysenteriae  Hiss,  B.  typhosus,  and  B. 
coli,  five  series  of  three  tubes  are  prepared.  The  first  set  is  inocu- 
lated with  B.  dysenteriae  Shiga,  the  second  with  B.  dysenteriae 
Flexner,  the  third  with  the  Hiss  strain,  and  the  fourth  and  fifth 
sets  with  B.  typhosus  and  B.  coli  respectively.  To  the  first  tube 
of  each  series  one  drop  of  the  filtrate  is  added,  to  the  second,  ten 


BACTERIOLYSIS  29 

drops,  and  to  the  third,  two  cubic  centimeters.  With  each  set 
the  procedure  is  that  already  indicated  for  the  Shiga  organism. 
In  routine  work  a  single  tube  can  be  used  in  place  of  the  three 
tubes,  and  to  this  ten  drops  of  the  filtrate  is  added,  but  with  this 
simplified  technic  the  danger  lies  in  the  fact  that  a  bacteriophage 
of  weak  activity  may  not  be  detected.3 

TECHNIC  FOR  ENHANCING  VIRULENCE 

It  has  already  been  stated  that  a  bacteriophage  may  be  present 
even  though  it  is  unable  to  induce  the  slightest  macroscopic  evi- 
dence of  the  lysis  of  a  bacterial  suspension.  Indeed,  this  is  the 
situation  most  frequently  encountered  in  the  process  of  isolation 
However,  it  is,  as  a  rule,  easy  to  increase  the  activity  of  such  a 
bacteriophage.  One  of  the  following  methods  suffices: 

3  Mention  may  be  made  here  of  a  method  of  Bordet  and  Ciuca,  and  it  is 
upon  this  procedure,  moreover,  that  these  authors  have  based  their  theory 
of  hereditary  lysis.  They  inoculate  a  guinea  pig  intraperitoneally  at  three 
or  four  different  times  at  intervals  of  a  few  days  with  a  culture  of  B.  coli. 
The  day  after  the  last  injection,  according  to  them,  it  is  only  necessary  to 
wash  out  the  peritoneal  cavity,  whereupon  the  principle  giving  rise  to 
"hereditary  lysis"  is  found  in  the  exudate.  From  their  first  communication 
on  the  subject  it  is  evident  that  they  consider  this  observation  a  specific 
example  of  a  general  law — which  indeed  would  be  one  of  the  sine  qua  non 
conditions  for  the  validity  of  their  theory — that  an  injection  of  any  bac- 
terium causes  the  organism  to  respond  with  the  production  of  a  principle 
which  gives  birth  to  the  phenomenon  of  lysis  in  series.  They  stated  that 
they  would  shortly  announce  the  results  secured  with  diverse  bacteria 
but  this  communication  has  never  appeared .  I  have  tried  without  success, 
as  have  several  other  investigators,  to  duplicate  the  results  described  by 
Bordet  and  Ciuca.  In  reality,  in  this  experiment,  there  has  been  a  passage 
of  the  anticoli  bacteriophage,  which,  as  experiment  shows,  is  normally 
present  in  the  guinea  pig  intestine,  into  the  peritoneal  cavity  as  a  result  of 
the  irritation  induced  by  the  inoculations.  After  its  appearance  in  the 
peritoneum  it  multiplies  there  by  virtue  of  the  B.  coli  inoculated.  The 
correctness  of  this  interpretation  is  vouched  for  by  the  fact  that  the  results 
reported  by  Bordet  and  Ciuca  can  be  secured  if,  a  few  hours  before  the 
intraperitoneal  injection  of  bacterial  culture,  the  bacteriophage  active 
for  this  bacterial  type  is  given  the  animal  per  os.  The  active  bacteriophage 
found  in  the  intestine  passes  through  into  the  peritoneal  cavity.  The 
method  of  Bordet  and  Ciuca  gives  results  only  by  accident,  only  when 
an  active  bacteriophage  was  previously  present  in  the  intestine.  Thus, 
all  of  the  conclusions  of  these  authors  fall  ipso  facto.  We  will  return  else- 
where to  this  question  (Chapter  VI,  Nature  of  the  Bacteriophage). 


30  THE   BACTERIOPHAGE 

When  the  agar  inoculation  has  shown  that  a  bouillon  suspension 
contains  an  active  bacteriophage  this  suspension  is  filtered  through 
infusorial  earth  and  then  through  a  bougie.  A  slightly  turbid 
suspension  is  prepared,  using  the  bacterial  strain  against  which 
the  bacteriophage  has  shown  some  activity,  and  into  this  suspen- 
sion are  introduced  some  four  or  five  drops  of  the  filtrate.  After 
an  incubation  period  of  twenty-four  hours  at  37°C.,  if  lysis  has 
not  been  produced,  this  second  bacterial  suspension  is  filtered 
as  before  and  a  third  suspension  is  inoculated  with  four  or  five 
drops  of  the  filtrate.  Such  transfers  are  continued  until  evident 
lysis  occurs.  During  the  process  it  is  easy  to  verify  the  presence 
of  the  bacteriophage  in  each  passage,  and  to  detect  any  increase 
in  virulence,  simply  by  spreading  the  successive  cultures  on  agar 
slants.  Comparison  of  the  cultures  secured  with  each  passage 
reflects  the  degree  of  virulence.  For  example,  the  agar  growth 
obtained  from  the  first  passage  shows  a  culture  growth  with  ten 
plaques,  the  second  passage  shows  100,  with  the  third  the  layer 
of  bacillary  growth  is  broken  up  with  an  abundance  of  the  areas, 
while  with  the  fourth  passage  only  a  few  isolated  colonies  of 
bacteria  are  seen.  It  can  be  readily  seen  that  the  virulence  of 
the  bacteriophage,  that  is,  its  ability  to  develop  at  the  expense  of 
the  bacteria,  increases  with  each  transfer  until  a  point  is  reached 
where  lysis  of  the  suspension  is  obtained. 

Successive  transfers  can  be  made  upon  agar  slants,  taking  the 
material  from  a  tube  showing  the  clear  areas.  With  a  platinum 
wire  material  can  be  removed  from  the  bacterial  growth  bor- 
dering on  a  plaque  and  inoculated  on  a  sterile  slant.  A  sec- 
ond, third,  and  fourth  (or  as  many  as  may  be  desired)  transfer 
from  agar  to  agar  can  be  made.  When  a  condition  is  reached 
where  the  agar  growth  shows  only  fragments  of  bacterial  culture 
the  surface  of  this  tube  is  carefully  washed  off  and  filtered  through 
infusorial  earth  and  a  bougie.  In  the  filtrate  is  found  a  bacterio- 
phage sufficiently  active  to  produce  lysis  of  a  bouillon  suspension. 

As  we  shall  see,  the  bacteriophage  is  not  destroyed  at  65°C., 
that  is,  at  a  temperature  above  the  thermal  death  point  of  most 
non-sporulating  bacteria.  Thus,  instead  of  filtration  the  appli- 
cation of  heat  may  be  employed.  Heating  at  58  to  60°C.  for 
thirty  minutes  will  kill  the  bacteria  and  not  harm  the  bacterio- 


BACTERIOLYSIS  31 

phage.  However,  filtration  has  always  appeared  to  give  more 
satisfactory  results.  In  a  later  chapter,  under  a  paragraph  en- 
titled "Multiple  Cultures"  a  third  procedure  will  be  considered. 

In  certain  cases  the  virulence  of  the  bacteriophage  can  be  in- 
creased in  vivo.  A  guinea  pig  is  injected  intraperitoneally  with 
two  cubic  centimeters  of  the  filtrate  containing  the  bacteriophage 
whose  virulence  it  is  desired  to  increase  and  with  a  few  cubic 
centimeters  of  the  bacterial  culture  against  which  the  bacterio- 
phage is  active.  After  twelve  to  eighteen  hours,  ten  cubic  centi- 
meters of  sterile  bouillon  is  injected  into  the  peritoneum  and 
a  few  minutes  later  the  peritoneal  exudate  is  removed  by  puncture 
with  a  trocar.  The  fluid  is  collected  in  a  few  cubic  centimeters 
of  citrate  solution  and  after  a  few  hours'  incubation  the  material 
is  filtered  (infusorial  earth  and  bougie) .  This  filtrate  frequently 
shows  that  a  bacteriophage  is  present  which  is  significantly 
more  virulent  than  that  which  was  introduced  into  the  guinea  pig. 

Usually  it  is  relatively  easy  to  increase  the  virulence  of  a  weak 
strain  of  the  bacteriophage,  but  at  times  it  may  become  very 
difficult,  particularly  when  working  with  strains  active  against 
the  Gram-positive  cocci.  In  such  cases  it  is  necessary  to  effect 
a  great  number  of  passages,  and  there  is  considerable  risk  of 
losing  the  bacteriophage  altogether,  particularly  during  the  first 
few  passages.  I  might  cite  as  an  example  an  anti-staphylococcic 
strain  with  which  Eliava  was  forced  to  make  passages  during 
four  months  in  order  to  obtain  sufficient  virulence  to  induce 
complete  lysis  of  a  suspension  containing  500  million  staphylo- 
cocci  per  cubic  centimeter. 

ENUMERATION  OF  THE  BACTERIOPHAGOUS  ULTRAMICROBES 

A  trace  of  a  filtrate  containing  a  bacteriophage  very  active 
for  a  given  bacterium  introduced  into  a  broth  suspension  of 
this  bacterium  causes  a  lysis  of  the  organisms  there  present  within 
a  few  hours.  The  medium  becomes  as  clear  as  broth  which 
has  never  been  inoculated.  A  trace  of  the  lysed  suspension  intro- 
duced into  a  new  suspension  similar  to  the  first  causes  a  similar 
lysis,  a  trace  of  this  second  lysed  suspension  introduced  into  a 
third  tube  reproduces  the  same  phenomenon,  and  so  on.  Dur- 
ing the  past  three  years  with  a  single  strain  daily  passages  have 


32  THE   BACTERIOPHAGE 

been  made,  sometimes  two  or  three  passages  on  a  single  day, 
introducing  in  each  transfer  about  0.001  cc.  of  the  last  tube  lysed 
into  a  fresh  suspension.  After  more  than  1500  passages  the  lysed 
suspension  of  the  last  tube  was  as  active,  even  more  active,  than 
the  filtrate  which  served  to  start  the  phenomenon  in  the  first 
tube  of  the  series. 

A  suspension  once  lysed  does  not  contain  any  living  bacteria. 
On  the  other  hand,  the  amount  of  bacteriophagous  ultrami- 
crobes  introduced  to  start  the  lysis  is  increased,  since  the  new 
lysate  is  as  active  as  was  the  preceding  one.  The  lysed  suspen- 
sion has  become  what  can  be  called,  literally,  a  culture  of  the 
bacteriophage. 

It  has  been  previously  stated  that  the  inoculation  of  agar  with 
a  bacterial  suspension  to  which  has  been  added  a  small  amount 
of  fluid  containing  the  active  principle  gives  a  bacterial  culture 
studded  with  clear  areas.  This  observation  suggests  a  means 
of  determining  with  approximate  exactness  the  phenomenon  of 
multiplication  of  ultramicrobes,  since  each  area  undoubtedly 
indicates  the  point  at  which,  during  the  inoculation,  there  was 
deposited  an  active  element, — an  ultramicrobe.  It  is  only  neces- 
sary to  work  with  measured  quantities  to  ascertain  the  number 
of  active  germs  in  a  fluid. 

From  an  abundance  of  experiments  a  single  one  showing  the 
method  of  counting  is  taken.  To  avoid  repetition  it  may  be 
stated  that  "suspension  of  B.  dysenteriae  Shiga"  is  to  be  under- 
stood. 

A  series  of  tubes  is  prepared,  once  for  all,  by  any  standard  pro- 
cedure, containing  B.  dysenteriae  Shiga  suspensions  of  the  follow- 
ing counts:  100,  200,  250,  300,  and  400  million  bacilli  per  cubic 
centimeter.  These  suspensions  are  stabilized  by  the  addition 
of  a  small  amount  of  formol  and  the  tubes  are  sealed  with  the 
blowpipe. 

The  suspension  to  be  subjected  to  lysis  should  be  taken  by 
preference  from  a  young  growth  on  agar.  A  concentrated  sus- 
pension is  prepared  by  adding  one  or  two  cubic  centimeters  of 
bouillon  to  the  agar  slant  and  allowing  the  tube  to  remain 
inclined  for  a  few  minutes  in  such  a  way  that  the  whole  bacterial 
growth  is  under  the  fluid.  With  shaking,  a  perfect  suspension 


BACTERIOLYSIS  33 

is  secured.  With  a  pipette  a  certain  quantity  of  this  concentrated 
suspension  is  added,  drop  by  drop,  into  a  tube  of  bouillon  until 
the  turbidity  corresponds  to  that  of  the  250  million  control  sus- 
pension. This  approximation  is  adequate  for  routine  practice, 
giving  a  suspension  which  contains  about  250  million  bacilli  per 
cubic  centimeter.  This  is  the  suspension  to  be  used.  A  young 
broth  culture  can  be  utilized,  but  the  other  suspension,  more 
accurately  adjusted,  is  to  be  preferred. 

Experiment  /.  To  a  tube  containing  10  cc.  of  a  suspension  of  B.  dysen- 
teriae  Shiga  is  added  0. 00,002  cc.  of  a  culture  of  the  bacteriophage  ten  days 
old,  i.e.,  a  Shiga  suspension  that  has  been  lysed  for  ten  days.  The  tube  is 
shaken  to  ensure  even  distribution  and  with  a  tared  platinum  loop  0.01  cc. 
of  the  liquid  is  removed  and  spread  as  uniformly  as  possible,  by  rubbing, 
over  the  surface  of  an  agar  slant.  This  tube  is  incubated  at  37°C.  After 
eighteen  hours  it  presents  a  Shiga  culture  studded  with  51  plaques. 

The  calculation  is  simple.  The  10  cc.  of  suspension  received 
0.00,002  cc.  of  the  bacteriophage  culture,  or  0.00,000,2  cc.  for 
each  cubic  centimeter  of  medium.  The  amount  of  material  taken 
for  planting  on  agar  was  0.01  cc.  This  contained,  therefore, 
0.00,000,002  cc.  of  the  original  bacteriophage  culture.  Upon  agar 
this  0.01  cc.  gave  51  clear  areas,  or  51  colonies,  each  one  of  which 
developed  from  a  single  bacteriophagous  germ  deposited  upon 
the  surface.  Fifty-one  germs  for  0.00,000,002  cc.  represent 
2,550,000,000  germs  per  cubic  centimeter.  This  is,  therefore,  the 
content  of  the  culture  of  the  bacteriophage  which  was  used  to 
inoculate  the  bacterial  suspension. 

The  technic  for  counting  the  ultramicroscopic  bacteriophage 
hardly  differs  from  that  used  in  counting  ordinary  bacteria. 
With  the  latter  isolated  colonies  are  secured  on  a  surface  other- 
wise sterile.  With  the  ultramicrobe,  clear  areas  representing 
colonies  are  obtained  superimposed  upon  a  layer  of  bacterial 
growth.  Counting  can  not  be  effected  otherwise,  since  the  bac- 
teriophagous ultramicrobe  is  only  able  to  develop  upon  the  bac- 
teria which  constitute  its  culture  medium. 

Each  plaque  represents  a  colony  of  the  bacteriophage.  This 
is  indisputable,  for  if  the  centre  of  such  an  area  is  touched  with 
the  point  of  a  drawn-out  capillary  pipette  and  this  pipette  is 
dropped  into  a  suspension  of  dysentery  bacilli,  the  culture,  when 


34  THE  BACTERIOPHAGE 

planted  upon  agar,  reveals  characteristic  clear  plaques.  If,  in 
the  bouillon,  lysis  is  allowed  to  proceed  for  some  hours,  there  are 
more  plaques.  The  plaque,  then,  to  all  appearances  sterile, 
is  in  reality  a  colony  of  the  bacteriophage. 

MULTIPLICATION   OF  THE   ULTRAMICBOSCOPIC    BACTERIOPHAGE 

The  multiplication  of  the  bacteriophage  in  the  course  of  its 
activity  can  be  followed  by  the  method  of  counting.  With  a 
definite  suspension  of  Shiga  bacilli,  always  250  million  per  cubic 
centimeter,  two  extreme  cases  are  very  interesting:  (1)  that 
which  occurs  when  a  large  number  of  ultramicrobes  are 
introduced,4  and  (2)  what  transpires  when  but  few,  or  only  a 
single  one,  is  inoculated. 

1.  Mass  inoculation.  Ten  cubic  centimeters  of  a  suspension 
of  Shiga  organisms  are  inoculated  with  0.04  cc.  of  the  culture  of 
the  bacteriophage.  The  culture  of  bacteriophage  contains  3000 
million  germs  per  cubic  centimeter.  Observed  macroscopically 
from  time  to  time,  it  will  be  seen  that  the  turbidity  gradually 
increases  up  to  about  the  third  hour,5  and  from  that  time  the 
liquid  clears  little  by  little.  Between  the  fourth  and  sixth  hours 
from  the  time  of  the  inoculation  the  suspension  has  become  limpid. 

4  As  already  stated,  study  of  the  bacteriophage  is  always  the  study  of  a 
complex  problem,  because  it  is  essential  to  consider  the  mutual  actions 
and  reactions  of  three  variable  factors — medium,  bacterium,  and  bacterio- 
phage. The  complexity  is  rendered  more  difficult  to  express  clearly  because 
of  the  lack  of  suitable  words.  Here,  for  example,  what  shall  we  term  the 
act  of  introducing  a  definite  quantity  of  the  bacteriophage  into  a  bacterial 
culture?  Obviously  the  term  "inoculation"  can  be  employed,  but  the 
medium  has  already  been  inoculated  with  the  bacterium;  thus  an  equivocal 
or  ambiguous  meaning  may  result .  The  proper  term  would  be  '  'contamina- 
tion" but  unfortunately  this  term  has  already  been  appropriated  in  bac- 
teriology as  a  synonym  for  "pollution".  The  term  "inoculation",  then, 
must  be  employed.  A  culture  medium  may  be  "seeded"  with  bacteria, 
and  then  "inoculated"  with  the  bacteriophage. 

•  Frequently  it  will  be  observed,  and  the  conditions  for  this  reaction 
are  not  exactly  determined,  that  the  dissolution  of  the  bacteria  is  preceded 
by  a  very  marked  agglutination.  This  reaction  is  noted  particularly 
when  the  bacterial  culture  is  inoculated  with  a  relatively  large  amount 
(twelve  drops  for  example)  of  a  bacteriophage  of  average  virulence. 


BACTERIOLYSIS  35 

If,  immediately  after  the  inoculation,  and  at  regular  intervals 
for  six  hours,  a  loopful  of  the  liquid  is  planted  on  agar,  all  the 
tubes  remain  sterile.  One  would  readily  think  that  the  absence 
of  colonies  of  Shiga  means  that  they  have  been  killed  with  the 
first  contact  with  the  inoculated  bacteriophage.  But  this  is 
not  the  case  for  if  instead  of  planting  agar  with  the  suspension 
as  such,  it  is  previously  diluted  to  1 : 1000  with  bouillon,  and  then 
this  dilution  is  planted  on  agar  numerous  colonies  of  B.  dysenteriae 
develop.  The  bacilli  have  by  no  means  been  killed.  If  the 
transfer  of  the  undiluted  suspension  to  agar  remains  sterile  it  is 
simply  because  the  agar  has  been  planted  simultaneously  with 
many  living  Shiga  bacilli  and  also  with  a  large  number  of  the 
ultramicrobes.  Indeed,  the  bacilli  have  commenced  to  multiply 
in  the  nutritive  medium  but  the  bacteriophage  has  not  been 
inactive.  Finding  the  bacilli  which  they  parasitize  within  reach, 
the  ultramicrobes  act  upon  them,  reproduce,  and  inhibit  the 
bacillary  growth.  In  the  other  case,  with  the  suspension  diluted 
to  1 : 1000,  the  bacilli  and  the  bacteriophagous  germ,  a  thousand 
times  less  numerous,  are  separated  by  spaces  sufficiently  great 
so  that  immediate  interaction  is  less  readily  accomplished.  This 
allows  the  bacilli  which  are  outside  of  the  immediate  neighborhood 
of  an  ultramicrobe  to  multiply  and  to  form  colonies. 

A  second  dilution  (1:1000)  of  the  suspension,  made  after  the 
latter  has  been  incubated  for  an  hour,  more  often  remains  sterile 
when  transferred  to  agar  and  only  rare  colonies  develop.  After 
two  hours  of  incubation,  cultures  on  agar  always  remain  sterile. 
At  this  time  all  the  bacilli  contained  in  the  suspension  have  been 
attacked  and  none  of  them  are  able  to  reproduce. 

2.  Inoculation  with  few  anti-microbial  elements.  Six  tubes  of 
the  Shiga  bacillus  suspension  are  inoculated  with  the  bacteriopha- 
gous culture  (containing  3000  million  per  cubic  centimeter)  in 
such  a  way  that  each  tube  receives  one  six-millionth  of  a  cubic 
centimeter.  When  incubated,  four  give  normal  cultures  of 
B.  dysenteriae  and  all  subcultures  on  agar  give  normal  growths. 
They  are  therefore  without  interest  to  us.  The  other  two,  how- 
ever, which  have  each  received  probably  one,  certainly  not  more 
than  two  ultramicrobes,  show  the  following  picture:  The  sus- 
pension becomes  more  and  more  turbid.  After  two  hours  at 


36  THE   BACTERIOPHAGE 

37°C.  the  opacity  is  about  two  times  as  great  as  at  the  beginning; 
after  three  hours,  it  is  about  two  and  one-half  times  as  great; 
after  four  hours,  about  three  times;  and  then  it  begins  to  diminish, 
so  that  after  five  hours  the  density  is  about  twice  as  great  as  at 
the  beginning  of  the  incubation.  This  clearing  continues  gradu- 
ally, so  that  after  fourteen  hours  the  culture  is  almost  entirely 
clear.  If  immediately  after  the  inoculation  with  the  bacterio- 
phage  and  then  every  thirty  minutes,  0.02  cc.  of  each  of  these 
two  suspensions  is  transferred  to  agar  slants,  these  tubes  will 
show,  after  incubation,  the  following: 

Plantings  after  one-half,  one,  one  and  one-half,  and  two  hours 
yield  normal  growths  of  B.  dysenteriae  Shiga.  After  two  and  one- 
half  hours  the  subcultures  show  three  plaques  in  one  tube,  five 
in  the  other  (average  four).  Therefore,  after  two  and  one-half 
hours  the  inoculated  suspension  contains  2000  bacteriophagous 
ultramicrobes. 

After  three  hours  the  tubes  show  five  and  four  respectively. 
Hence,  there  has  been  no  material  increase  between  two  and 
one-half  and  three  hours. 

The  three  and  one-half  hour  plantings  show  nine  and  five 
areas  (average  seven).  The  number  of  bacteriophagous  ele- 
ments has  slightly  increased. 

After  four  hours,  the  agar  tubes  show  101  and  111  plaques  re- 
spectively (average  105).  After  four  hours,  therefore,  the  num- 
ber of  ultramicrobes  is  between  fifty  and  sixty  thousand. 

After  four  and  one-half  hours,  the  counts  are  145  and  160 
(average  152),  indicating  that  the  suspension  contains  75,000; 
a  number  but  slightly  different  from  the  count  after  four  hours. 

After  five  hours,  the  agar  tubes  are  sterile.  When  diluted  to 
1 : 1000  in  a  suspension  of  Shiga  bacilli  and  transferred  immediately 
to  agar  in  the  same  way,  the  tubes  give  four  and  six  areas.  Thus, 
it  appears  that  after  five  hours  the  suspension  contains  about 
1,500,000  bacteriophagous  germs. 

From  this  it  is  readily  apparent  that  the  multiplication  of 
the  ultramicrobes  is  extremely  rapid,  and,  what  is  most  remark- 
able, it  is  associated  with  successive  jumps,  each  augmentation 
being  separated  by  an  interval  of  about  seventy-five  minutes. 
We  will  refer  elsewhere  to  this  experiment  when  we  consider  the 
mode  of  reproduction  of  the  bacteriophage. 


BACTERIOLYSIS  37 

This  experiment  shows  that  it  is  only  necessary  to  have  a  single 
bacteriophage  present  in  a  bacterial  suspension  to  produce  a 
complete  lysis  of  the  bacteria,  provided  the  strain  of  bacteriophage 
is  of  maximum  activity. 

Needless  to  say,  such  experiments  have  been  repeated  many 
times,  always  with  results  comparable  to  those  cited.  Indeed, 
this  statement  holds  for  all  of  the  experiments  reported  in  this 
monograph — all  have  been  repeated. 

The  attention  of  investigators  should  be  called  to  this  particu- 
lar point,  namely,  that  once  a  strain  of  bacteriophage  of  suffi- 
cient virulence  has  been  obtained,  the  end  results  of  the  experi- 
ments are  always  the  same,  without  exception — an  increase  in 
the  number  of  ultramicrobes  inoculated  and  a  complete  lysis 
of  the  bacterial  suspension.  Repeating  the  same  experiment 
several  times  with  the  same  strain  of  bacteriophage,  employing 
always  the  same  conditions  of  medium  and  temperature,  the 
proliferation  of  the  ultramicrobe  progresses  in  the  same  manner 
and  lysis  is  effected  in  the  same  length  of  time.  But  if  one  is 
making  a  comparative  study  of  different  strains,  although  all 
are  endowed  with  high  activity,  certain  differences  are  noted. 
With  one  strain  complete  lysis  will  be  obtained  after  three  and 
one-half  hours  (this  is  the  shortest  period  thus  far  observed), 
with  another  only  after  fourteen  hours,  all  the  conditions  being 
the  same.  In  a  word,  and  this  observation  likewise  applies  to 
all  of  the  experiments  here  reported,  the  phenomenon  always 
proceeds  as  has  been  indicated.  The  time  alone  may  vary.  The 
ultramicroscopic  bacteriophage  is  a  living  being,  and  as  such, 
the  processes  which  it  carries  out  can  not  go  on  with  the  regularity 
of  a  diastatic  action. 

Experiment  II.  Here  is,  to  cite  an  example,  an  experiment  conducted 
with  another  strain  of  bacteriophage.  It  will  be  noted  that  lysis  is  effected 
much  more  quickly  than  in  the  instance  given  above.  The  suspension  of 
Shiga  bacilli  is  made  in  bouillon  previously  warmed  to  38°C.,  and  the 
suspension  is  inoculated  with  0.00,01  cc.  of  the  culture  of  bacteriophage. 
The  macroscopic  appearance  showed  that : — After  two  hours  the  suspension 
is  three  times  as  turbid  as  at  first. 

After  two  and  one-half  hours  it  is  about  three  and  one-half  times  as 
turbid. 

After  two  and  three-quarter  hours  it  is  about  three  times  as  turbid  as  at 
.first. 

After  three  hours  the  turbidity  is  hardly  apparent. 


38  THE   BACTERIOPHAGE 

In  this  experiment  the  lysis  was  almost  entirely  accomplished 
within  a  space  of  fifteen  minutes,  that  is,  during  the  period  of 
time  between  two  and  three-quarters  and  three  hours  after  the 
inoculation. 

A  single  ultramicroscopic  bacteriophage  is,  therefore,  adequate 
to  provoke  lysis.  If  successive  dilutions  of  a  culture  of  the  bac- 
teriophage are  prepared,  one  drop  of  the  culture  into  a  tube  of 
sterile  bouillon,  one  drop  of  this  first  dilution  into  a  second  tube, 
a  drop  of  the  second  into  a  third,  and  so  on,  and  if  into  each  of 
these  dilutions  a  fixed  quantity  of  a  concentrated  Shiga  culture 
is  introduced,  lysis  is  secured  in  all  tubes  which  have  received  at 
\  least  one  ultramicrobe.  This  is  usually  the  first  four  tubes  of  the 

series.  The  remaining  tubes  will  show  a  normal  growth  of  the 
Shiga  bacillus.  Since  we  have  been  able  to  make  counts  of  the 
bacteriophage  and  recognize  the  rapidity  with  which  even  a  single 
ultramicrobe  can  proliferate  and  bring  about  lysis,  these  observa- 
tions are  self-explanatory.  Without  this  means  of  investigation 
one  woulpl  be  liable  to  commit  a  serious  error  and  to  conclude 
that  the  sterile  bouillon  of  the  first  four  tubes  had  contained  a 
culture  of  the  bacteriophage  other  than  that  introduced  in  pre- 
paring the  dilutions.  Incidentally,  this  error  has  been  committed 
by  certain  authors.  In  reality,  while  there  has  been  a  dilution, 
the  diluted  culture  was  active  just  so  long  as  there  was  to  be 
found  a  single  ultramicrobe. 

Recognizing  the  number  of  ultramicrobes  in  a  lysed  suspension, 
which  has,  in  effect,  become  a  culture  of  the  bacteriophage,  and 
the  value  of  the  dilution,  it  can  be  mathematically  determined 
whether  a  bacterial  suspension  inoculated  with  such  a  dilution 
will  undergo  lysis  or  not.  This  test  has  been  performed  by  ex- 
periment more  than  a  hundred  times  with  very  diverse  strains 
of  the  bacteriophage. 

THE  BACTERIOPHAGE:  AN  OBLIGATORY  PARASITE 

Whatever  may  be  the  medium  employed,  in  the  absence  of  a 
bacterium  for  which  the  bacteriophage  is  active,  multiplication 
of  the  ultramicrobes  never  takes  place.  And  this  remains  true 
even  if  inoculated  into  a  medium  containing,  instead  of  living 
bacteria,  organisms  that  have  been  killed  by  any  procedure  what- 


BACTERIOLYSIS  39 

soever.  All  experiments  have  been  uniformly  negative  in  attempt- 
ing to  obtain  multiplication  of  the  bacteriophage  by  contact  of 
the  ultramicrobe  with  bacteria  killed  by  age,  by  heat,  by  chloro- 
form, by  thymol,  by  the  essences  of  cinnamon  and  mustard,  by 
alcohol,  by  bichloride  of  mercury,  by  phenol,  by  sulfuric  and 
hydrochloric  acids,  and  by  iodine.  With  such  suspensions  no 
action  whatever  is  secured; — no  lysis  and  no  culture  of  ultrami- 
crobes. 

The  bacteriophage  is  an  obligatory  parasite,  multiplying  only 
at  the  expense  of  living  bacteria. 

An  experiment  of  the  following  nature  is  interesting  in  that 
it  shows  that  the  bacteriophage  will  attack  only  normal  bacteria. 
The  bacteria  are  suspended  in  a  medium  containing  an  antiseptic 
in  a  quantity  so  small  that  the  bacteria  will  only  be  killed  after  a 
time  exceeding  that  required  for  the  lytic  process  of  the  bacte- 
riophage. In  such  a  case  the  bacteria  are  not  affected  by  the 
bacteriophage  and  the  latter  fail  entirely  to  'proliferate.  The 
antiseptic  selected  was,  by  intention,  one  without  action  on  the 
diastases, — sodium  fluoride. 

In  a  one  per  cent  solution  of  sodium  fluoride  in  bouillon  the  Shiga 
bacilli  are  still  cultivable  after  thirty-six  hours,  a  time  more 
than  adequate  for  the  bacteriophage  to  manifest  its  lytic  activ- 
ity and  to  multiply.  Furthermore,  if  transfers  in  series  are  made 
with  such  a  suspension,  inoculating  the  first  tube  with  a  drop  of 
the  bacteriophage  culture;  the  second,  after  incubation  for  twenty- 
four  hours,  with  a  drop  of  the  first;  the  third  with  a  drop  of  the 
second,  and  so  on,  it  will  be  found  that  the  bacteriophage  disap- 
pears with  the  transfer  from  the  second  to  the  third  tubes  of  the 
series  (this  can  be  confirmed  by  placing  a  drop  of  each  tube  into  a 
suspension  of  normal  bacilli).  Controlling  this  procedure  with 
a  second  series,  using  pure  bouillon  or  even  sterile  water,  it  will 
be  found  that  here  likewise  the  bacteriophage  will  not  be  present 
in  the  third  or  fourth  tube.  This  is,  as  will  be  seen  later,  simply 
a  result  of  dilution.  The  bacteriophagous  germ,  therefore,  can 
not  be  cultivated  in  a  suspension  containing  fluoride,  in  sterile 
bouillon,  or  in  pure  water. 

Moreover,  it  has  been  shown  that  it  is  solely  because  of  the 
medium  into  which  it  is  introduced  that  the  bacillus  is  not  sub- 


40  THE   BACTERIOPHAGE 

ject  to  attack.  For  after  a  twenty-four  hour  stay  in  fluoride 
bouillon  a  normal  culture  will  be  secured  by  transfer  from  this 
medium  into  ordinary  bouillon,  and  this  culture  is  normally 
lysed  by  the  bacteriophage. 

This  will  be  discussed  later,  accepting  for  the  moment  that  here 
is  a  medium  in  which  the  Shiga  organism  remains  alive  for  at 
least  thirty-six  hours,  in  which  the  bacteriophage  likewise  remains 
alive,  which  exerts  no  inhibitory  action  on  the  diastases,  but  in 
which  the  bacteriophage  fails  to  multiply. 

THE  EFFECT  OF  THE  CONDITION  OF  THE  BACTERIA 

These  experiments  have  been  conducted  so  as  to  determine  the 
effect  of  the  state  of  the  bacterium  upon  the  lytic  process.  Since 
lysis  is  the  result  of  the  multiplication  of  the  ultramicrobes  the 
lytic  process  will  not  be  complete  unless  all  of  the  bacteria  present 
in  the  suspension  are  capable  of  being  attacked. 

Instead  of  taking  a  suspension  prepared  from  a  young  culture 
we  may  inoculate  the  bacteriophage  into  a  fifteen-day  old  broth 
culture.  A  clearing  of  the  medium,  a  partial  lysis,  results  but  a 
certain  degree  of  turbidity  remains.  Nevertheless,  it  is  possible 
to  continue  to  use  such  a  medium,  making  as  many  passages  as 
may  be  desired.  Some  tube  of  the  series  when  planted  on  agar 
or  in  bouillon  will  remain  sterile,  and  a  drop  of  this  tube  inoculated 
into  a  suspension  of  young  bacilli  will  produce  perfect  lysis.  In 
the  old  culture,  then,  the  bacteriophage  multiplies  normally 
but  does  not  produce  lysis,  or  at  least,  the  lysis  is  incomplete. 
What  is  the  explanation  of  this  reaction?  To  answer  this  it  is 
sufficient  to  compare  the  results  of  counting  the  total  number  of 
bacilli  existing  in  an  old  culture  (this  can  be  done  by  the  method 
of  counting  cells)  with  the  results  secured  by  counting  the  viable 
organisms  only  (done  by  the  plating  method).  For  a  confirma- 
tion of  this  type,  a  Shiga  culture  in  Martin's  bouillon  is  made, 
incubated  for  fourteen  hours,  and  allowed  to  stand  at  laboratory 
temperature  for  fifteen  days.  The  total  count  of  bacillary  bodies 
will  be  about  625  millions;  that  of  the  viable  bacilli,  that  is,  those 
capable  of  yielding  colonies  when  transferred  to  agar,  will  be 
about  two  millions  in  each  half  cubic  centimeter  of  culture.  Now, 
as  we  have  seen,  the  bacteriophage  is  only  able  to  develop  at  the 


BACTERIOLYSIS  41 

expense  of  living  bacteria,  these  being  the  ones  which  are  lysed. 
In  the  old  suspension  which  we  have  mentioned  in  which  there 
is  only  about  one  organism  in  three  hundred  which  is  capable  of 
being  dissolved,  it  can  readily  be  comprehended  that  if  lysis  of  a 
suspension  be  taken  as  a  criterion,  the  bacteriophage  appears  to 
be  without  action  in  such  a  culture. 

It  is  useless  to  work  with  old  cultures.  In  a  broth  culture  of 
Shiga,  after  only  twenty-four  hours  of  incubation,  as  has  been 
shown,  about  one-third  of  the  organisms  present  are  incapable  of 
producing  colonies  when  planted  on  agar.  If,  on  the  other  hand, 
an  agar  slant  culture  is  utilized,  almost  all  of  the  bacteria  are  liv- 
ing after  twenty-four  hours  at  37°C.  A  twenty-four  hour  bouillon 
culture  will,  then,  remain  slightly  turbid  when  the  lytic  process 
is  accomplished,  while  a  suspension  made  in  broth  from  a  young 
agar  culture  containing  the  same  number  of  bacteria  will  be 
perfectly  limpid  when  the  lysis  is  achieved.  In  this  last  case 
all  of  the  bacteria  were  living  and  susceptible  to  the  attack  of  the 
bacteriophage.  It  is  for  this  reason  that  it  is  preferable  to  effect 
cultures  of  the  bacteriophage  in  a  suspension  rather  than  directly 
into  a  bouillon  culture. 

Certain  bacteria  give  a  homogeneous  growth  in  a  young  cul- 
ture in  bouillon  but  when  taken  from  agar  they  can  be  suspended 
only  with  difficulty.  B.  pestis  is  such  an  organism.  When  work- 
ing with  such  bacteria  it  is  preferable  to  have  the  bacteriophage 
act  on  a  broth  culture  in  the  following  manner.  A  bouillon  tube 
is  lightly  inoculated  with  the  bacterium.  When  the  culture  has 
clouded,  the  bacteriophage  active  for  this  bacterial  strain  is 
introduced  and  at  the  same  time  the  culture  is  diluted  with  an 
equal  volume  of  sterile  medium.  This  dilution  should  be  made 
before  the  bacteriophage  has  had  time  to  multiply  sufficiently 
to  parasitize  an  appreciable  number  of  bacteria.  Thus,  the 
bacterial  culture  at  the  time  of  " departure,"  that  is,  when  the 
bacteriophagous  organisms  are  sufficiently  abundant,  will  consist 
almost  entirely  of  young  bacilli,  readily  subject  to  attack. 

It  has  been  demonstrated  that  the  products  of  bacterial  growth 
as  found  in  an  old  culture,  products  which,  as  is  well-known, 
inhibit  the  development  of  bacteria  (as  in  the  so-called  "vac- 
cinated" media)  are  without  effect  upon  the  lytic  phenomenon. 


42  THE  BACTERIOPHAGE 

Experiment  III.  Cultures  of  B.  dysenteriae  Shiga,  aged  fifteen  days 
and  eighteen  hours  respectively,  are  centrifugalized.  The  sediment  from 
the  first  culture  is  suspended  in  the  supernatant  fluid  of  the  second,  and  the 
sediment  of  the  second  culture  in  the  fluid  of  the  first.  Both  suspensions 
thus  formed  are  inoculated  with  a  drop  of  a  culture  of  the  bacteriophage. 
The  suspension  consisting  of  "old"  bacilli  and  "young"  medium  remains 
turbid ;  that  of  "young"  bacilli  and  "old"  medium  becomes  perfectly  limpid 
after  seven  hours. 

Thus,  while  the  products  of  bacterial  metabolism  are  not  in- 
hibitory for  the  lytic  process,  the  products  of  lysis,  as  we  will 
see,  exert  quite  a  different  action.  These  products  are  the  result 
of  the  activity  of  the  ultramicroscopic  bacteriophage,  and  as 
such,  they  impede  its  activity. 

THE   INFLUENCE   OF   THE  MEDIUM 

It  is  evident  from  the  foregoing  experiments  that  the  true  cul- 
ture medium  of  the  bacteriophage  is  the  living  bacterium.  The 
nature  of  the  fluid  in  which  the  bacteria  are  suspended  is  without 
direct  influence  upon  the  culture  of  the  bacteriophage,  provided 
only  the  bacteria  remain  living  in  it  throughout  a  sufficient  period 
of  time  and  provided  it  does  not  alter  the  constitution  of  the 
bacterial  cell.  Experiment  confirms  this  statement. 

The  only  additional  condition  regarding  the  medium  is  that  it 
be  alkaline  in  reaction.  Lysis  will  not  take  place  in  a  medium 
of  acid  reaction.6 

Experiment  IV.  Peptone  water  (containing  25  grams  of  Chassaing  pep- 
tone and  5  grams  of  NaCl  per  liter)  is  neutralized  to  phenolphthalein.  The 
medium  is  then  frankly  alkaline  to  litmus.  It  is  then  distributed  into 
tubes,  10  cc.  to  each.  Hydrochloric  acid  is  added  to  each  tube  in  dilutions 
to  form  an  increasing  scale  of  acidity.  All  of  the  tubes  are  inoculated  with 
a  concentrated  suspension  of  Shiga,  sufficient  to  yield  a  normal  suspension 
of  250  millions  per  cubic  centimeter.  Finally,  each  tube  is  inoculated  with 
0.001  cc.  of  a  culture  of  the  bacteriophage.  After  twenty-four  hours  the 
numbers  of  bacteriophage  in  the  several  tubes  are  determined  by  the 
method  previously  described. 

6  Certain  commercial  peptones  contain  glucose  in  appreciable  quantity, 
hence  their  use  may  be  attended  by  failure. 


BACTERIOLYSIS 


43 


TUBE 

REACTION  TO 
PHENOLPHTHALEIN 

APPEARANCE  OF  THE 
SUSPENSION  AFTER 
TWENTY-FOUR  HOURS 

NUMBER  OP  ULTRA- 
MICROBES  PER  CUBIC 
CENTIMETER 

1 

0 

Very  slight  clouding 

400,  000,  000 

2 

-  2 

Very  slight  clouding 

500,  000,  000 

3 

-  4 

Limpid 

500,  000,  000 

4 

-  6 

Limpid 

1,250,000,000 

5 

-  8 

Limpid 

2,750,000,000 

6 

-10 

Limpid 

1,000,000,000 

7 

-12 

Limpid 

1,  000,  000,  000 

8 

-14 

Slight  turbidity 

250,  000,  000 

9 

-16 

Turbid 

500,  000 

10 

-18 

Turbid 

1,000,000 

11 

-20 

Turbid 

500,  000 

12 

-22 

Turbid 

None 

Neutrality  to  litmus  corresponds  to  about  —  16,  thus  it  may 
be  concluded  that  the  bacteriophage  ceases  to  grow  when  the 
medium  presents  even  the  slightest  acidity.  The  ultramicrobial 
elements  detected  in  the  slightly  acid  tubes — those  where  lysis 
is  not  produced — is  not  an  indication  of  multiplication.  They 
are  simply  the  ultramicrobes  which  were  inoculated,  still  living. 
When  the  medium  is  decidedly  acid  even  these  elements 
are  destroyed. 

The  following  experiment  demonstrates  this  still  better  and 
further  confirms  the  fact  that  the  bacteria  constitute  the  only 
culture  medium  for  the  bacteriophage.7 

7  As  will  be  shown  in  a  later  chapter,  bacteria  in  general  are  destroyed 
after  exposure  for  a  very  short  time  in  physiological  saline.  On  the  other 
hand,  the  different  strains  of  dysentery  bacilli  present  a  variable  resistance. 
Certain  races  are  no  longer  cultivable  in  bouillon  after  an  exposure  of  five 
to  six  hours,  others  remain  cultivable  up  to  45  to  50  days.  Experiments 
involving  lysis  in  physiological  saline  ought  to  be  performed  with  strains 
of  the  last  type.  Moreover,  it  is  necessary  to  employ  an  extremely  active 
bacteriophage,  capable  of  exerting  its  action  in  the  minimum  length  of 
time — if  possible,  one  capable  of  producing  complete  lysis  in  about  three 
hours.  A  saline  suspension  of  a  bacterial  strain  of  weak  vitality  may  be 
already  sterile  after  four  to  five  hours,  that  is  to  say,  the  great  majority  of 
the  bacteria  are  dead  before  the  lytic  process  is  able  to  be  effected .  And  the 
bacteriophage  is  without  action  on  dead  bacteria. 


44  THE   BACTEBIOPHAGE 

Experiment  V.  One  hundred  cubic  centimeters  of  a  suspension  of  young 
Shiga  bacilli  are  prepared  in  0.85  per  cent  saline,  neutral  to  litmus.  This 
suspension  is  inoculated  with  five  drops  of  an  earlier  saline  culture  of  the 
bacteriophage,  and  the  material  is  divided  into  five  portions,  20  cc.  in  each 
tube.  The  first  of  these  remains  as  already  prepared.  The  second  is  ren- 
dered alkaline  by  the  addition  of  NaOH  in  a  quantity  sufficient  to  give  an 
alkalinity  equivalent  to  10  mgm.  of  NaOH  per  liter.  The  remaining  tubes 
are  also  rendered  alkaline,  the  third  having  a  reaction  equal  to  25  mgm.  of 
NaOH  per  liter,  the  fourth  equal  to  50  mgni.,  and  the  fifth  equal  to  100  mgm. 
After  incubation  for  eighteen  hours  the  first  tube  shows  its  original  tur- 
bidity, tube  2  is  cloudy,  and  tubes  3,  4,  and  5  are  almost  perfectly  clear. 
In  these  the  turbidity  is  such  that  it  can  just  be  detected  and  is  due  to  the 
fact  that  a  certain  number  of  the  bacilli  had  died  in  the  saline  before  they 
were  attacked.  When  the  lysis  is  completed  in  the  last  three  tubes,  to  tube 
1,  which  maintained  its  original  turbidity,  NaOH  is  added  in  an  amount 
equal  to  100  mgm.  per  liter.  Twelve  hours  later  lysis  has  taken  place; 
the  suspension  has  been  transformed  so  that  it  possesses  a  transparency 
comparable  to  tube  5.  It  has  been  demonstrated  that  this  degree  of 
alkalinity  by  itself  is  without  effect  on  the  Shiga  bacilli,  since  they  develop 
normally  in  bouillon  containing  as  much  as  500  mgm.  of  soda  per  liter. 
From  the  cleared  tubes  all  subcultures  remain  sterile. 

The  bacteriophage  can  be  indefinitely  cultured  in  series  in  a 
slightly  alkaline  saline  solution.  Indeed,  the  salt  CNaCl)  itself 
can  be  dispensed  with.  Serial  cultures  have  been  maintained 
in  chemically  pure  water  containing  only  25  mgm.  of  soda  per  liter. 

Certain  experiments  with  a  synthetic  medium,  capable  of  main- 
taining a  culture  of  the  Shiga  bacillus,  were  performed  and  are 
not  without  interest.  Growth  of  the  dysentery  bacilli  and  com- 
plete lysis  by  the  bacteriophage  are  secured  in  a  medium  of  the 
following  composition:  water,  80  cc.;  sodium  chloride,  1  cc.; 
potassium  phosphate,  1  cc.;  sodium  phosphate,  1  cc.;  and  aspara- 
gine,  3  cc.,  all  in  10  per  cent  solutions.  The  medium  is  rendered 
alkaline  in  accordance  with  the  nature  of  the  experiment  to  be 
performed. 

In  bouillon  a  relatively  high  alkalinity  does  not  interfere  with 
lysis. 

Experiment  VI.  Five  tubes,  each  containing  10  cc.  of  bouillon  ren- 
dered alkaline  to— 8  are  seeded  with  a  suspension  of  the  Shiga  bacillus  and 
then  inoculated  with  0.02  cc.  of  a  culture  of  the  bacteriophage.  A  N/10 
solution  of  NaOH  is  added;  to  the  second  tube  0.5  cc.,  to  the  third  1  cc., 
to  the  fourth  1 .5  cc.,  and  to  the  last  2  cc.  The  first  four  tubes  are  perfectly 
lysed  after  eighteen  hours,  only  the  fifth  remains  clouded. 


BACTERIOLYSIS  45 

It  may  be  affirmed,  therefore,  that,  aside  from  the  matter  of 
alkalinity,  the  composition  of  the  medium  with  respect  to  its 
nutritive  properties,  exerts  no  influence  on  the  development  of 
the  bacteriophage.  From  the  moment  when  it  has  at  its  disposi- 
tion living  and  normal  bacterial  cells,  against  which  it  is  active, 
it  multiplies — at  the  expense  of  these  bacteria  which  constitute 
its  sole  culture  medium. 

CULTURE     OF    THE    BACTERIOPHAGE     ON     SOLID    MEDIA!   ISOLATED 

COLONIES 

It  has  been  stated  that  the  bacteriophage  shows  the  formation 
of  obvious  colonies  on  agar,  and  that  in  order  to  obtain  them  it 
is  only  necessary  to  inoculate  a  broth  suspension  of  the  Shiga 
organism  very  lightly  with  a  culture  of  the  bacteriophage  and  to 
distribute  a  drop  of  this  suspension  on  agar.  After  incubation 
the  covering  of  bacillary  growth  presents  a  number  of  areas  free 
of  all  apparent  culture.  If  the  inoculation  of  the  bacteriophage 
has  been  massive  the  surface  of  the  agar  appears  sterile.  Let  us 
consider  the  characters  of  these  cultures. 

When  the  surface  of  the  agar  remains  bare  because  of  the  large 
number  of  bacteriophagous  organisms  and  maintains  this  appear- 
ance indefinitely  it  has  become  unsuited  for  the  cultivation  of 
the  Shiga  bacillus.  When  inoculated  at  such  a  time  with  a  cul- 
ture of  this  bacillus,  even  in  a  very  abundant  sowing,  the  slightest 
development  cannot  be  detected.  The  medium  is,  however, 
normal  for  another  bacterium.  If  inoculated  with  the  cholera 
vibrio,  for  example,  the  growth  will  be  as  luxuriant  as  if  planted 
upon  fresh  medium.  Hence,  if  B.  dysenteriae  Shiga  does  not  grow 
it  is  only  because  the  bacteriophagous  organisms  remain  on  the 
surface  of  the  agar  and  exercise  their  dissolving  action  on  the 
bacteria  deposited  thereon.  This  is  readily  confirmed.  If  we  take 
a  tube  of  agar  which  has  remained  apparently  sterile  after  having 
been  inoculated  with  a  suspension  of  the  bacteria  containing  a 
bacteriophagous  culture,  and  if  the  surface  of  the  medium  in  such 
a  tube  is  washed  with  a  few  drops  of  sterile  bouillon  and  to  this 
is  added  a  fresh  suspension  of  bacteria,  this  suspension  will  be 
lysed  within  a  few  hours. 


46  THE   BACTERIOPHAGE 

It  sometimes  happens,  especially  when  using  agar  somewhat 
dried  out,  that  a  few  colonies  of  Shiga  are  obtained,  always  lo- 
cated at  the  extreme  edge  of  the  layer  of  agar.  We  will  return 
to  this  extremely  interesting  particular  in  the  discussion  of  sec- 
ondary cultures. 

If,  instead  of  a  continuous  covering  of  the  bacteriophagous 
growth  the  ultramicrobes  are  deposited  in  limited  areas — and 
this  is  readily  accomplished  by  placing  drops  of  culture  on  the 
sterile  surface  of  a  tube  of  agar,  or  again,  by  drawing  lines  over 
the  surface  with  a  platinum  loop  dipped  in  the  culture  of  bacterio- 
phage,  and  after  the  tubes  have  remained  inclined  for  a  few  hours 
in  the  incubator  to  secure  drying — we  find  that  the  areas  impreg- 
nated with  the  bacteriophagous  culture  remain  free  of  Shiga 
bacilli,  but  that  these  organisms  grow,  on  the  contrary,  perfectly 
well  on  the  parts  not  covered  by  the  bacteriophage. 

When  in  the  suspension  planted  upon  agar  the  number  of 
bacilli  is  infinitely  great  and  the  number  of  the  ultramicrobes  is 
sufficiently  small,  the  bacteriophage  culture  as  individual  units 
is  distributed  over  the  surface  of  the  agar,  and  under  such  cir- 
cumstances the  bacterial  layer  will  appear  studded  with  apparently 
sterile  areas.  These  areas,  or  plaques,  have  a  circular  form  with 
a  diameter  of  from  1  to  5  mm.  The  plaques  are  in  general  of  the 
greatest  extent  when  the  suspension  is  somewhat  weak  although 
sufficiently  concentrated  to  give  a  continuous  layer  of  growth 
rather  than  isolated  colonies.  On  such  a  tube  the  areas  are 
larger  as  the  subjacent  medium  becomes  thicker,  that  is,  toward 
the  bottom  of  the  tube.  Upon  a  Petri  dish,  where  the  agar  layer 
is  of  essentially  the  same  thickness  throughout,  all  of  the  plaques 
of  a  given  culture  are  of  approximately  the  same  diameter.  As 
will  be  seen,  the  area  of  the  plaque  bears  a  relationship  to  the 
virulence  of  the  bacteriophage  which  causes  it. 

If  a  tube  or  plate  presenting  plaques  is  held  in  the  incubator 
at  37°C.,  or  at  an  entirely  different  temperature,  no  change 
occurs  in  the  plaques;  their  diameter  remains  indefinitely  what 
it  was  at  first.  They  are  never  covered  or  encroached  upon  by 
the  bacterial  culture.  At  no  time  does  there  exist  within  the 
extent  of  the  plaque,  whatever  its  size  may  be,  microscopically 
visible  bacterial  cells.  The  plaque  is  always  rigorously  sterile. 


BACTERIOLYSIS  47 

As  soon  as  the  culture  is  well  developed,  as  after  18  to  24  hours 
of  incubation,  if  .the  centre  of  such  a  plaque  is  touched  with  a 
platinum  wire  and  this  is  immersed  in  a  culture  of  Shiga  bacilli 
the  bacteriophage  develops  in  this  suspension  and  the  latter  is 
lysed  after  a  few  hours.  The  plaque,  although  sterile,  is  not 
ultrasterile;  it  is  in  fact  a  colony  of  the  bacteriophage. 

Furthermore,  if  a  trace  of  the  bacillary  growth  at  the  periphery 
of  a  plaque  is  taken  with  a  platinum  wire  and  seeded  on  agar  it 
remains  sterile  and  inoculation  into  a  bacterial  culture  shows  that 
the  bacteriophage  is  present  there  also.  But  when  the  bacillary 
layer  is  taken,  not  at  the  immediate  edge  of  the  area,  but  at  a 
distance  of  two  millimeters  from  it,  for  example,  and  planted,  the 
tubes  show  the  growth  of  a  normal  culture.  The  bacteriophage 
is  not  found. 

If  the  culture  showing  the  plaques  is  returned  to  the  incubator 
and  the  tests  are  repeated  three  or  four  days  later,  that  is,  culturing 
the  bacillary  growth  at  a  distance  of  two  millimeters  from  a 
plaque  onto  agar  and  into  a  suspension  it  will  be  found  that  the 
bacteriophage  is  there  present  at  that  time.  The  bacteriophage  has, 
therefore,  gradually  invaded  the  bacillary  layer.  This  invasion  is 
always  slow — proceeding  more  and  more  slowly  as  time  pro- 
gresses— so  that  the  ring  invaded,  even  after  several  months, 
amounts  to  a  zone  but  a  few  millimeters  wide.  Beyond  the  limits 
of  this  zone  the  Shiga  organisms  remain  cultivable  just  as  long 
as  they  do  in  a  normal  control  culture  without  the  bacteriophage. 

The  question  immediately  arises  as  to  why  the  bacteriophage 
does  not  invade  the  entire  layer  of  bacterial  growth.  For  this 
there  are  two  reasons.  The  bacteriophage  attacks  the  bacterial 
cell  most  readily  when  the  bacterium  is  young.  When  placed  upon 
agar  the  bacteriophagous  organisms  find  themselves  located 
in  the  immediate  vicinity  of  bacilli  which  reproduce  actively  as 
soon  as  they  are  deposited  upon  a  nutrient  medium.  They  find 
then,  within  their  range,  very  young  bacilli  distributed  in  a  very 
thin  layer  over  the  agar.  Lysis  is  thus  possible  and  the  apparent 
sterility  of  the  plaque  results.  But  beyond  this  zone  invaded  by 
the  bacteriophage  during  the  first  few  hours  the  bacilli  develop 
freely  forming  a  layer  of  increasing  thickness  comprised  of  or- 
ganisms of  increasing  age.  In  other  words,  a  thicker  and  thicker 


48  THE   BACTERIOPHAGE 

layer  of  bacilli  always  becoming  more  and  more  resistant  to  lysis 
develops.  This  can  be  readily  demonstrated  by  direct  experi- 
mental proof. 

If  the  agar  surface  in  a  Petri  dish  is  heavily  seeded  with  a 
Shiga  culture  and  at  some  point  on  this  a  drop  of  the  culture  of 
the  bacteriophage  is  placed,  and  after  a  three-hour  incubation 
period  another  drop  of  the  bacteriophage  is  placed  on  the  surface 
and  this  same  process  repeated  after  six,  twelve  and  twenty  hours, 
with  continuous  incubation  of  the  plate  during  the  intervals,  it 
will  be  found  fifteen  hours  later  that  the  areas  upon  which  the 
first  three  drops  were  placed  have  remained  sterile — no  bacillary 
growth  has  taken  place.  At  the  point  where  the  fourth  drop 
was  placed,  that  is,  after  the  culture  had  been  incubated  for  twelve 
hours,  there  is  a  thin  layer  of  growth  composed  of  dead  bacilli. 
The  area  where  the  drop  of  bacteriophage  was  placed  after  twenty 
hours  presents  an  appearance  practically  normal.  These  five 
spots,  then,  represent  the  diverse  aspects  of  an  isolated  colony 
of  the  bacteriophage,  as  from  the  centre  to  the  periphery. 

The  second  reason  is  of  a  more  general  nature,  representing  a 
phenomenon  common  to  the  majority  of  cultivable  organisms. 
The  colonies  of  the  bacteriophage  act  absolutely  like  colonies 
of  those  bacteria  which,  except  for  organisms  such  as  B.  proteus, 
never  progressively  invade  the  surface  of  solid  media.  Thus, 
if  the  Shiga  bacillus  is  inoculated  upon  agar  in  an  amount  suitable 
to  yield  isolated  colonies,  after  18  to  24  hours,  each  colony  will 
be  from  two  to  four  millimeters  in  diameter,  the  largest  colonies 
to  be  found  at  the  points  where  the  medium  has  the  greatest 
depth,  that  is,  toward  the  bottom  of  the  tube.  Such  colonies 
increase  in  size  but  very  slowly,  always  more  and  more  slowly  as 
time  progresses,  and  even  after  two  months,  the  zone  of  increase 
will  not  be  greater  than  a  few  millimeters.  From  the  bacteriologi- 
cal point  of  view  it  is  not  peculiar,  as  has  been  suggested,  that 
the  bacteriophage  does  not  invade  the  entire  bacterial  layer. 
It  must  be  borne  in  mind  that  the  bacteriophage,  far  from  being 
dissimilar  to  other  cultivable  organisms,  behaves,  when  in  iso- 
lated colonies,  exactly  like  a  colony  of  bacteria. 

Why  does  not  the  bacterial  colony  continue  to  increase  in  size 
and  to  invade  the  entire  surface  of  the  medium?  Because  the 


BACTERIOLYSIS  49 

soluble  substances  resulting  from  the  vital  activity  of  the  bac- 
teria diffuse  into  the  agar  and  these  substances  constitute  an 
actual  specific  antiseptic  which  limits  the  culture.  The  medium 
is  "  vaccinated"  around  the  colony.  The  deeper  the  agar  layer, 
or  the  farther  the  colonies  are  separated,  the  greater  the  volume 
of  the  substratum  capable  of  diluting  this  antiseptic  substance,  and 
the  larger  will  be  the  colony.  The  situation  is  precisely  the  same 
with  the  bacteriophage;  the  more  scattered  the  colonies  and  the 
deeper  the  substratum,  the  greater  the  diameter.  Direct  ex- 
perimentation proves  the  correctness  of  this  interpretation,  and 
that  the  soluble  substances  elaborated  during  the  lytic  process, — 
substances  resulting  from  the  vital  activity  of  the  bacteriophage, 
— inhibit  the  vital  processes,  delay  growth,  and  prevent  the  ac- 
complishment of  total  lysis. 

EFFECT    OF    THE    CONCENTRATION    OF  BACTERIA  IN   THE   MEDIUM; 
INHIBITORY   EFFECTS   OF  THE   PRODUCTS   OF  LYSIS 

In  all  of  the  experiments  involving  the  action  of  the  bacterio- 
phage in  a  liquid  medium  which  we  have  up  to  the  present  con- 
sidered the  bacterial  suspension  has  contained  approximately 
250,000,000  organisms  per  cubic  centimeter. 

What  occurs  if  the  concentration  of  suspended  bacilli  is  varied 
between  50  and  500  millions?  The  end  result  will  always  be 
the  same — a  complete  lysis — and  even  the  time  required  for  this 
lysis  will  not  greatly  vary  for  the  inoculation  of  a  given  quantity 
of  the  bacteriophagous  culture,  such  is  the  rapidity  of  develop- 
ment of  these  organisms.  Let  us  consider  the  two  extreme  cases. 

Experiment  VII  (A.)  The  inoculation  with  the  bacteriophage  is 
massive,  that  is,  0.02  cc.  In  such  a  case  the  difference  in  time  is  most 
marked. 

Number  of  bacilli  Lysis  is  complete 

per  cc.  in 

50,000,000 4|  hours 

100,000,000 4£  hours 

200,000,000 5    hours 

250,000,000 5    hours 

300,000,000 5£  hours 

400,000,000 6    hours 

500,000,000 8   hours 

(B.)  The  inoculation  with  the  bacteriophage  is  very  weak,  in  the  follow- 
ing experiment,  0.00,000,1  cc. 


50  THE   BACTERIOPHAGE 

Number  of  bacilli  Lysis  is  complete 

per  cc.  in 

50,000,000 14*  hours 

100,000,000 14*  hours 

200,000,000 14*  hours 

250,000,000 14*  hours 

300,000,000 16   hours 

400,000,000 16   hours 

500,000,000 18   hours 

These  results  are  readily  understood.  In  the  first  case  the 
number  of  ultramicrobes  is  very  great  from  the  start,  all  of  the 
bacteria,  or  by  far  the  greater  part  of  them,  are  immediately 
attacked,  and  this  quickly  arrests  the  development  of  the  bac- 
terial culture.  In  the  second  case,  as  a  result  of  the  small  num- 
ber of  ultramicrobes  inoculated,  very  few  of  the  bacteria  are  at 
once  attacked,  and  those  which  remain  unharmed  are  free  to 
develop,  so  much  the  more  as  the  suspension  is  the  more  dilute. 
To  be  convinced  of  this  it  is  only  necessary  to  observe  the  sus- 
pensions and  to  compare  their  relative  opacity  from  time  to  time. 
All  become  more  and  more  turbid  during  the  first  few  hours  after 
the  inoculation,  and  in  five  or  six  hours  after  the  inoculation  they 
all  present  a  comparable  opacity,  corresponding  to  approximately 
650,000,000  bacilli  per  cubic  centimeter.  In  a  word,  whatever 
may  be  the  original  titre  of  the  suspension  at  the  time  when  it 
is  inoculated  with  a  limited  number  of  the  bacteriophagous  or- 
ganisms, the  latter  must  always  operate  on  a  suspension  of  about 
650,000,000  bacilli  per  cubic  centimeter,  since  in  all  cases  the 
bacteria  reproduce  until  they  attain  this  number.  Hence, 
lysis  will  always  take  place  within  very  nearly  the  same  length 
of  time. 

A  suspension  of  young  bacilli,  containing  about  500,000,000 
bacilli  per  cubic  centimeter  is  completely  lysed  by  the  action  of 
a  bacteriophage  of  maximum  activity.  Beyond  this  figure  the 
medium  never  entirely  clears,  and  remains  the  more  cloudy  the 
more  concentrated  the  suspension,  regardless  of  the  number  of 
ultramicrobes  inoculated.  A  suspension  containing  1,000,000,000 
bacilli  per  cubic  centimeter,  for  example,  will  never  be  completely 
lysed,  whether  it  is  inoculated  with  a  few  individual  bacteriophag- 
ous organisms  or  whether  it  is  inoculated  with  some  thousands  of 
millions.  However,  in  working  with  suspensions  which  are  ex- 


BACTERIOLYSIS 


51 


tremely  heavy  it  is  found  that  the  bouillon  transplants  made  on 
to  agar  after  eighteen  to  twenty-four  hours  are  always  sterile. 
The  bacilli  have  been  killed  but  not  completely  lysed. 

Experiment  VIII 


SUSPENSION  OF  BACILLI 
(MILLIONS  PER  CUBIC 
CENTIMETER) 

INOCULATED  WITH  CULTURE 
OF  BACTERIOPHAGE 

ASPECT  OF  SUSPENSION  AFTER 
TWENTY-FOUR  HOURS 

CC. 

5,000 

0.1 

Turbid 

2,000 

0.1 

Cloudy 

1,000 

0.1 

Slightly  cloudy 

500 

0.1 

Clear 

After  incubation  for  8  hours  all  cultures  appeared  the  same. 

The  inhibitory  force  which  interferes  with  lysis  is  due  to  the 
accumulation  of  the  soluble  products  resulting  from  the  lytic 
process,  that  is,  to  the  activity  of  the  ultramicroscopic  bacterio- 
phage  itself.  In  this  respect  the  action  of  the  bacteriophage 
is  in  accord  with  a  phenomenon  common  to  all  cultivable  micro- 
organisms. 

Experiment  IX.  A  bouillon  suspension  containing  250,000,000  bacilli 
per  cubic  centimeter  is  inoculated  with  0 . 001  cc .  of  a  culture  of  the  bacterio- 
phage. The  next  morning,  or  after  fourteen  hours,  lysis  is  complete.  A 
count  of  the  bacteriophage  shows  that  there  are  1,600,000,000  per  cubic 
centimeter.  At  this  time  a  concentrated  bacterial  suspension  is  added 
to  the  lysed  suspension  in  such  concentration  that  the  titre  amounts  to 
250,000,000  per  cubic  centimeter.  Seven  hours  later  the  medium  is  again 
limpid,  and  a  count  shows  the  presence  of  2,100,000,000  ultramicrobes. 
This  second  lysis  completed,  the  bacterial  content  is  again  restored.  This 
time  lysis  is  hardly  accomplished  in  48  hours,  indeed,  at  this  time  the 
bouillon  is  not  quite  clear.  A  count  gives  2,400,000,000  ultramicrobes  per 
cubic  centimeter.  At  this  time,  then,  the  medium  contains  in  each  cubic 
centimeter  the  dissolved  substance  of  750,000,000  bacilli.  The  suspen- 
sion is  made  up  to  a  concentration  of  250,000,000  once  more  (for  the  fourth 
time).  Eight  hours  later  the  culture  has  cleared  somewhat  but  remains 
decidedly  cloudy.  The  count  shows  2,600,000,000  ultramicrobes.  Inocula- 
tions upon  agar  and  into  broth  remain  sterile. 

It  is  plainly  to  be  seen,  therefore,  that  the  more  concentrated 
the  medium  becomes  in  dissolved  substances  the  more  marked 
becomes  the  inhibition  and  the  less  active  the  culture  of  the 
bacteriophage. 


52  THE   BACTERIOPHAGE 

The  quantity  of  the  bacteriophagous  germs  inoculated  into 
the  suspension  is  without  influence  on  the  final  result. 

Experiment  X.  The  following  tubes  containing  suspensions  of  the  Shiga 
bacillus  are  prepared. 

Tubes  1  and  5  contain  250  million  per  cubic  centimeter 
Tubes  2  and  6  contain  500  million  per  cubic  centimeter 
Tubes  3  and  7  contain  1000  million  per  cubic  centimeter 
Tubes  4  and  8  contain  2000  million  per  cubic  centimeter 

Tubes  1,  2,  3,  and  4  are  inoculated  with  0.001  cc.  of  a  culture  of  the  bacte- 
riophage.  Tubes  5,  6,  7,  and  8  are  inoculated  with  0. 5  cc.  of  the  same  culture 
of  bacteriophage,  or,  500  times  as  much  as  in  the  first  set.  After  eight 
hours  tubes  1  and  5  are  limpid.  After  fourteen  hours  tubes  1,  2,  5,  and 
6  are  limpid.  After  four  days  tubes  1,  2,  5,  and  6  are  still  limpid,  tube  3  is 
very  slightly  cloudy,  tubes  4  and  7  are  cloudy,  and  tube  8  is  turbid. 

It  is  thus  apparent  that  lysis  is  not  affected  by  the  number  of 
the  ultramicrobes  inoculated.  We  will  see  in  a  subsequent 
chapter  which  treats  of  the  resistance  of  the  bacteria  to  the 
bacteriophage,  that  contrary  to  what  one  would  a  priori  suppose, 
lysis  of  a  culture  is  even  more  perfect  when  the  amount  of  the 
bacteriophage  added  to  the  suspension  is  rather  small. 

Quite  aside  from  the  quantitative  relationships,  a  suspension 
may  vary  in  "  quality.'7  One  may  work  with  bacteria  of  different 
ages,  or  with  organisms  of  different  strains.  In  so  far  as  differ- 
ence in  strains  is  concerned,  the  course  of  the  phenomenon  remains 
essentially  the  same — at  least  in  so  far  as  the  Shiga  bacillus  is 
concerned.  We  shall  see  that  it  is  not  the  same  with  certain  other 
bacteria,  B.  typhosus  for  example. 

With  reference  to  the  question  of  the  age  of  the  bacilli  subjected 
to  the  action  of  the  bacteriophage  we  have  already  seen  that  the 
younger  the  bacillus  the  more  readily  it  is  attacked.  This  dif- 
ference is  due  solely  to  the  state  of  the  bacillus  itself  and  not  to 
the  soluble  substances  resulting  from  its  activity; — such  substances 
as  result  in  a  "  vaccination"  of  the  medium,  to  use  a  common  ex- 
pression. The  bacteria  vaccinate  the  medium  for  themselves 
through  the  products  of  their  activity.  The  bacteriophage  does 
the  same  thing.  But  the  soluble  products  resulting  from  their 
respective  activities  have  nothing  in  common. 


BACTERIOLYSIS  53 

EFFECT   OF   EXTERNAL   PHYSICAL   CONDITIONS 

The  presence  or  absence  of  oxygen  has  no  effect  upon  the  course 
of  the  phenomenon.  The  rapidity  of  multiplication  of  the  ultra- 
microbes  and  the  duration  of  the  lytic  process  are  the  same  in 
aerobiosis  as  in  anaerobiosis. 

On  the  other  hand,  as  would  naturally  be  expected,  the  effect 
of  the  temperature  is  marked. 

Experiment  XI.  Three  tubes  containing  a  suspension  of  the  Shiga  bacil- 
lus are  inoculated,  each  tube  receiving  0.00,000,01  cc.  of  the  bacteriophage 
culture.  These  tubes  are  placed  at  different  temperatures;  the  first  at  8°, 
the  second  at  22°,  and  the  third  at  37°C. 

The  suspension  held  at  8°C.  shows  no  lysis  after  twenty-four  hours,  but 
after  15  to  16  days  lysis  is  complete.  The  number  of  ultramicrobial  ele- 
ments at  this  time  is  180,000,000  per  cc.  as  compared  with  20  to  25  per  cc. 
when  they  were  introduced. 

The  suspension  kept  at  22°C.  shows  that  multiplication  commences  after 
three  hours.  At  this  time  the  count  is  75  ultramicrobes  per  cubic  centi- 
meter. After  five  hours  the  count  is  8,000,  after  eight  hours  190,000,  and 
at  twenty-five  hours  lysis  is  complete  and  the  count  is  780,000,000  per  cubic 
centimeter. 

The  suspension  held  et  37°C.  shows  210  ultramicrobes  after  two  hours, 
10,000  after  three  and  one-half  hours,  200,000  after  five  hours,  and 
1,700,000,000  per  cubic  centimeter  after  thirteen  hours,  with  a  complete 
lysis. 

Between  37  and  41°C.  the  course  of  the  reaction  does  not  show 
appreciable  variation.  Between  41  and  44°C.  the  lysis  is  less 
and  less  complete.  For  this  there  are  two  reasons;  the  develop- 
ment of  the  ultramicrobes  is  less  and  less  active,  and  the  number 
of  bacilli  killed  in  consequence  of  the  elevation  of  temperature 
is  greater  and  greater.  As  a  result  the  number  of  organisms  ca- 
pable of  being  attacked  and  dissolved  are  less  and  less  numerous. 
However,  serial  cultivation  of  the  bacteriophage  at  44°C.  is  still 
possible.  The  ultramicrobe  can  be  cultivated  at  temperatures 
higher  than  those  supported  by  B.  dysenteriae.  With  the  latter 
growth  ceases  at  43°C. 

In  effect,  the  optimum  temperature  for  the  bacteriophage  is 
the  same  as  that  for  the  bacterium,  as  is  but  logical,  since  all 
experimental  work  demonstrates  that  the  more  closely  the  bac- 
terial cell  approaches  normal  so  much  the  better  is  it  attacked. 


54  THE   BACTERIOPHAGE 

EFFECT  OF  ANTISEPTICS  UPON  LYSIS 

To  complete  the  macroscopic  study  of  the  phenomenon  it  may 
be  well  to  consider  what  effect  different  substances  that  may  be 
added  to  the  suspension  may  have  upon  the  lytic  process. 

As  we  will  see  with  reference  to  the  properties  of  the  bacterio- 
phage,  although  it  does  not  present  a  resistance  as  marked  as 
some  of  the  ultramicrobes  to  chemical  and  physical  destructive 
agents,  it  is,  nevertheless,  less  susceptible  than  the  majority  of 
cultivable  organisms. 

From  the  particular  point  of  view  of  lysis,  we  must  recall  that 
the  action  of  antiseptics  is  complex.  The  bacteriophage  is  only 
able  to  grow  at  the  expense  of  living  bacteria  and  all  action  ex- 
erted on  the  bacteria  of  a  suspension  are  reflected  in  the  phenom- 
enon of  lysis,  even  if  these  actions  are  weak  or  wholly  lacking 
upon  the  bacteriophage  itself. 

The  special  resistance  of  the  bacteriophage  to  antiseptics  does 
not  modify  the  lytic  process. 

Antiseptic  substances  may  be  introduced  into  the  suspension 
in  amounts  sufficiently  weak  to  render  their  effect  on  the  bac- 
teria negligible  and  thus  fail  to  alter  the  course  of  the  phenomenon. 
If,  on  the  contrary,  the  amount  of  antiseptic  is  such  that  it  is 
capable  of  recognition,  the  bacteriophage  may  be  unable  to  multi- 
ply for  lack  of  normal  bacteria  and  lysis  is  prevented.  In  this 
last  case  it  is  easy  to  see  that  the  conditions  of  the  experiment 
are  the  same  as  if  the  bacteriophage  was  placed  in  the  presence  of 
bacteria  previously  modified  by  the  antiseptic  in  question.  And 
it  has  already  been  shown  that  under  these  conditions  neither  the 
growth  of  the  bacteriophage  nor  the  lysis  resulting  therefrom  is 
accomplished. 

The  experiment  previously  presented  showed  that  the  bac- 
teriophage failed  to  multiply  in  bouillon  suspensions  of  Shiga  con- 
taining one  per  cent  of  sodium  fluoride,  even  though  the  bacteria 
remained  alive  during  a  period  of  time  amply  sufficient  for  com- 
plete lysis  to  be  effected  in  the  presence  of  normal  bacteria. 
Glycerine  acts  in  a  different  manner.  In  high  concentrations 
this  substance  prevents  the  growth  of  the  bacteria,  but  its  activity 
is  inhibitory  rather  than  strictly  antiseptic. 


BACTEKIOLYSIS  55 

Experiment  XII.  Tubes  of  bouillon,  containing  glycerine  in  the  fol- 
lowing concentrations,  are  seeded  with  a  drop  of  an  eighteen-hour  bouillon 
culture  of  B.  dysenteriae  Shiga.  The  results  secured  with  the  different  con- 
centrations are: 

Bouillon  +    5  per  cent  of  glycerine :  Very  abundant  growth 
Bouillon  +  10  per  cent  of  glycerine :  Weak  growth 
Bouillon  +  15  per  cent  of  glycerine :  Very  slight  growth,  with  sedi- 
ment 
Bouillon  +  20  per  cent  of  glycerine :  Clear  medium,   with   abundant 

sediment  of  bacteria 

Bouillon  +  25  per  cent  of  glycerine :  Clear  medium,  slight  sediment 
Bouillon  +  30  per  cent  of  glycerine :  Clear  medium,  slight  sediment 
Bouillon  +  35  per  cent  of  glycerine :  Clear  medium,  trace  of  sediment 
Bouillon  +  40  per  cent  of  glycerine :  No  growth  whatever 
All  subcultures  made  from  these  tubes  at  the  end  of  forty-eight  hours 
give,  in  normal  bouillon,  normal  growths. 

B.  typhosus  is  somewhat  more  sensitive  to  the  action  of  glycerine.  Even 
in  a  medium  containing  10  per  cent  the  growth  is  insignificant. 

Bacteria  suspended  in  glycerine  bouillon,  even  in  a  concentra- 
tion of  25  per  cent,  remain  alive  for  at  least  forty-eight  hours; 
that  is,  throughout  a  time  amply  sufficient  for  the  bacteriophage 
to  develop  and  to  effect  lysis,  as  was  the  case  with  the  fluoride 
medium.  But  the  following  experiments  show  that  the  culture 
of  the  bacteriophage  in  the  glycerine  medium  is  absolutely  nor- 
mal while  it  is  entirely  lacking  in  the  fluoride  medium. 

I  emphasize  the  fact  of  the  growth  of  the  bacteriophage  and 
the  lysis  which  is  the  result  of  this  growth  in  a  glycerine  medium, 
for  it  will  be  necessary  to  return  to  these  experiments  when  we 
review  the  various  proofs  concerning  the  living  nature  of  the 
bacteriophage. 

Experiment  XIII.  Tubel.  Prepare  a  suspension  of  B.  dysenteriae 
Shiga,  250,000,000  per  cubic  centimeter,  in  bouillon  containing  35  per  cent 
of  glycerine.  Inoculate  with  0.02  cc.  of  the  bacteriophage  culture.  Nor- 
mal lysis  occurs  in  eight  hours.  A  control  suspension  of  the  Shiga  bacilli 
in  the  glycerinated  medium,  but  without  the  bacteriophage,  yields  positive 
subcultures  up  to  the  seventh  day. 

Tube  2.  Shiga  bacilli,  250,000,000  per  cubic  centimeter,  are  suspended 
in  bouillon  with  50  per  cent  glycerine.  The  medium  is  inoculated  with 0. 02 
cc.  of  the  bacteriophage.  Normal  lysis  takes  place  in  ten  hours.  The 
control  suspension,  without  the  bacteriophage,  is  cultivable  up  to  the 
forty-eighth  hour. 


56  THE   BACTERIOPHAGE 

Tube  3.  The  Shiga  bacilli,  in  the  same  concentration,  are  suspended  in 
bouillon  with  60  per  cent  of  glycerine.  Portions  of  this  suspension  are 
inoculated  with  various  amounts  of  the  bacteriophage  culture,  as  follows: 

a.  With  0.5  cc.  of  the  bacteriophage  culture.  With  this  amount  lysis 
is  complete  in  eight  hours. 

6.  With  0. 02  cc.  of  the  bacteriophage  culture.  Complete  lysis  is  obtained 
in  nine  hours. 

c.  With  0.0001  cc.  of  the  bacteriophage  culture.  No  lysis  results.  The 
ultramicrobes  inoculated,  too  few  in  number,  have  not  had  time  to  develop 
before  the  bacilli  are  dead.  A  control  suspension,  without  the  bacterio- 
phage, gave  positive  cultures  only  up  to  the  18th  hour. 

From  this  it  appears  that  in  glycerine  broth  the  bacilli  remain 
normally  susceptible  to  attack  by  the  bacteriophage  just  as  long 
as  they  are  living. 

Although  the  bacteria  die  when  suspended  in  the  glycerine 
medium  it  can  not  be  assumed  that  the  glycerine  acts  as  a  true 
antiseptic,  that  is,  that  it  modifies  the  bacterial  protoplasm.  No 
one  would  contend  that  sodium  chloride  in  weak  concentration  is 
an  antiseptic  despite  the  fact  that  non-spore-forming  bacteria 
suspended  in  normal  saline  survive  for  but  a  very  short  time: 
twenty-four  to  forty-eight  hours  in  the  case  of  the  Shiga  bacillus. 

The  experiments  on  lysis  in  the  glycerinated  media  are,  more- 
over, of  great  interest  in  that  glycerine,  in  very  high  concentra- 
tions, sterilizes  cultures  of  the  bacteriophage. 

Substances  without  action  on  the  bacterial  cells  are,  in  general, 
without  influence  on  lysis.  This,  for  example,  is  the  case  with 
normal  serum,  ascitic  fluid,  urine,  and  2.5  per  cent  sodium  chloride. 
Calcium  chloride,  on  the  other  hand,  has  a  very  marked  inhibi- 
tory effect,  and  potassium  chlorate  delays  lysis.  In  low  concen- 
trations magnesium  sulfate  and  the  phosphates  of  sodium  and 
potassium  favor  lysis.  This  is  particularly  observed  in  the  case 
of  strains  of  the  bacteriophage  of  feeble  activity. 

When  sugars  which  are  not  fermented  by  the  bacterium  against 
which  the  bacteriophage  is  active  are  added  to  the  suspension 
lysis  is  not  modified.  The  addition  of  fermentable  sugars  is 
without  effect  if  the  inoculation  of  the  bacteriophage  is  massive, 
but  if  the  inoculation  is  weak  lysis  does  not  occur  or  remains 
incomplete,  depending  upon  the  amount  inoculated. 


BACTERIOLYSIS  57 

The  cause  for  these  results  is  very  obvious.  The  bacteriophage 
is  very  sensitive  to  acidity  and  with  a  minimal  inoculation  the 
bacteria  begin  to  develop,  to  attack  the  sugar,  and  to  render 
the  medium  acid  before  the  ultramicrobes  are  present  in  ade- 
quate numbers  to  effect  lysis  in  the  time  at  their  disposal. 

SOLUBLE    SUBSTANCES    OF   THE    BACTERIA 

Lysis  of  the  bacteria  by  the  bacteriophage  is  complete,  with- 
out residue.  In  the  last  analysis  this  lysis  can  only  be  in  the 
nature  of  a  diastatic  action.  No  bacterial  activity  is  caused 
simply  by  the  presence  of  the  organisms  as  such,  but  rather  by 
virtue  of  the  secretory  products  which  they  elaborate.  The  ul- 
tramicrobes necessarily  secrete  some  of  these  lytic  diastases — the 
lysins — which  liquefy  the  substances  constituting  the  bacterial 
body.  This  point  will  be  considered  further.  The  chemical 
aspect  of  the  reaction  has  been  investigated  but  little  since  such 
studies  naturally  fall  within  the  field  of  the  chemist.  All  that 
may  here  be  stated  is  that  whatever  may  remain  in  solution  in 
the  clear  medium  when  lysis  is  complete,  it  is  not  protein  in 
nature,  as  is  indicated  by  the  reaction  to  heating. 

The  viscosity  of  a  suspension  containing  500,000,000  Shiga 
bacilli  per  cubic  centimeter  differs  from  that  of  the  bouillon  used 
in  preparing  the  suspension.  A  volume  of  bouillon  giving  nor- 
mally 100  drops  gives  but  97  when  the  suspension  is  lysed. 

The  decomposition  of  the  substances  derived  from  the  bacterial 
bodies  continues  certainly  for  some  time  after  the  lysis.  This 
can  be  shown  for  the  Shiga  bacteriolysate  by  the  fact  that  if  a 
rabbit  is  inoculated  with  the  material  immediately  after  the 
lysis  is  completed,  it  is  killed  by  a  dose  essentially  the  same  as  the 
minimal  lethal  dose  of  the  suspension.  The  toxicity  of  the  bac- 
teriolysate falls  very  rapidly;  a  week  after  the  lysis  it  is  markedly 
diminished,  and  in  fifteen  days  the  material  is  practically  non- 
toxic.  On  the  other  hand,  it  is  well  known  that  the  Shiga  endo- 
toxin  is  very  stable.  Obviously  then,  this  endotoxin  is  rapidly 
destroyed  by  the  bacteriophage.  In  a  later  chapter  we  shall 
see  that  the  lysin  secreted  by  the  bacteriophage  can  be  obtained 
by  precipitation  with  alcohol. 


58  THE  BACTERIOPHAGE 

THE  ULTRAMICROBIAL  BACTERIOPHAGE:  AN  ENDOPARASITE 

We  have  already  considered  the  mode  of  action  of  the  bacterio- 
phage  from  the  point  of  view  of  its  macroscopic  characteristics. 
Various  types  of  experiment  allow  us  to  penetrate  somewhat  further 
into  the  more  intimate  nature  of  the  phenomenon. 

Experiment  has  demonstrated  that  the  bacteriophage  is  ca- 
pable of  development  only  at  the  expense  of  living  bacteria,  since 
these  provide  its  sole  culture  medium.  This  cultivation  appar- 
ently takes  place  within  the  interior  of  the  bacterial  cell,  and 
ultramicroscopic  observation  shows  that  this  is  indeed  the  case. 
But  first,  let  us  consider  some  of  the  experiments  which  permit  us 
to  recognize  the  manner  in  which  the  infection  of  the  bacteria 
is  accomplished. 

Attempts  have  been  made  to  effect  a  culture  of  the  bacterio- 
phage of  the  Shiga  bacillus  in  a  filtrate  derived  from  Shiga  or- 
ganisms grown  in  bouillon  for  various  lengths  of  time — 1,  7,  14, 
and  21  days — but  all  have  failed.  Two  counts  of  the  number  of 
ultramicrobial  elements,  the  one  made  immediately  after  the 
inoculation,  the  other  after  incubation  for  a  week  at  37°C., 
have  given  exactly  the  same  number  of  germs.  Thus,  the  bac- 
teriophage is  entirely  incapable  of  multiplication  in  a  medium 
containing  only  the  secretory  products  of  the  bacterium.  The 
bacterial  body  itself  is  essential. 

If  the  bacteriophage  actually  proliferates  within  the  interior 
of  the  bacterial  cell,  the  ultramicrobes  inoculated  into  a  suspension 
ought,  before  all  multiplication,  to  disappear  from  the  fluid.  In 
fact,  each  ultramicrobe  ought  first  to  penetrate  a  bacterial  cell, 
to  multiply  there  and  to  reappear  in  the  fluid  only  when  this  cell 
is  destroyed.  It  is  easy  to  verify  this  hypothesis. 

Experiment  XIV.    The  following  suspensions  are  prepared : 

(1)  100  cc.  of  a  suspension  of  the  Shiga  organisms  containing  250,000,000 
bacilli  per  cubic  centimeter.    This  is  inoculated  with  0. 25  cc.  of  a  culture 
of  the  bacteriophage. 

(2)  100  cc.  of  a  suspension  of  the  cholera  vibrio,  containing  250,000,000 
per  cubic  centimeter.    This  also  is  inoculated  with  0.25  cc.  of  the  same 
culture  of  anti-Shiga  bacteriophage. 

(3)  100  cc.  of  bouillon  containing  only  0.25  cc.  of  the  same  bacterio- 
phage. 


BACTEKIOLYSIS  59 

The  material  of  all  three  flasks  is  incubated  at  37°C.  Immediately  after 
the  inoculation,  after  thirty  minutes,  and  again  after  1  hour,  20  cc.  are 
taken  from  each  of  the  three  flasks  and  centrifuged  at  4,000  revolutions  per 
minute  for  ten  minutes. 

There  are  thus  nine  tubes  which  have  been  centrifuged.  From  the 
supernatant  fluid  of  each  of  these  0.02  cc.  is  taken  and  introduced  into 
other  tubes  containing  suspensions  of  the  Shiga  bacillus,  and  the  counts 
of  the  ultramicrobe  are  made  by  plating  0. 02  cc.  of  each  of  these  nine  tubes 
on  six  plates  of  medium.  In  this  way  an  average  of  the  counts  can  be  ob- 
tained. The  results  of  these  counts  indicate  the  number  of  ultramicrobes 
remaining  in  the  medium,  since  those  which  have  penetrated  the  bacterial 
cells  before  the  centrifugation  have  been  thrown  down  with  the  cells  during 
this  procedure  and  in  consequence  are  to  be  found  in  the  sediment. 

The  results  of  the  counts  are  as  follows : 

Tube  1.    Shiga  suspension  plus  bacteriophage. 

a.  Counts  of  the  material  made  immediately  after  the  preparation  are 
214,  193,  187,  221,  229,  and  183  plaques.  The  average  is  204,  representing 
5,000,000  germs  per  cubic  centimeter  in  the  original  suspension  immediately 
after  inoculation. 

6.  Counts  on  the  suspension  after  incubation  for  30  minutes  are  3,  7, 
4,  6,  6,  and  3  plaques.  The  average  is  5.  This  indicates  that  there  are 
125,000  bacteriophagous  germs  in  the  suspension  thirty  minutes  after  the 
inoculation.  That  is  to  say,  of  each  41  ultramicrobes  inoculated  40  have 
disappeared. 

A  direct  count  of  the  suspension  without  centrifugation  gives 
5,000,000,000  elements  per  cubic  centimeter.  It  is  therefore  certain  that 
the  ultramicrobes  which  have  disappeared  from  the  fluid  during  the  cen- 
trifugation have  gone  down  with  the  bacteria.  And,  as  we  will  see  in  the 
two  control  experiments,  in  the  absence  of  Shiga  bacilli  this  sedimentation 
of  the  bacteriophage  does  not  occur  (at  least,  when  centrifuged  at  a  speed 
of  4000  revolutions). 

c.  After  1  hour,  the  count,  made  as  before  on  the  supernatant  fluid 
gives  an  average  of  8  plaques,  or  200,000  ultramicrobes  per  cubic  centimeter, 
a  number  essentially  the  same  as  that  secured  after  thirty  minutes.    At 
this   time   a   count   of   a  suspension   which  has  not  been   centrifuged 
gives  6,500,000,  a  number  very  close  to  that  secured  immediately  after  the 
inoculation. 

It  should  be  noted  that  in  the  hypothesis  formulated  with  regard  to 
intrabacterial  growth,  each  colony  forming  within  the  interior  of  a  bac- 
terium gives  rise  to  but  a  single  plaque ;  just  as  in  a  bacterial  count  made  by 
the  same  method  a  whole  clump  of  bacteria  seeded  upon  agar  will  yield 
only  a  single  colony. 

d.  Counts  made  upon  the  suspension  with  and  without  centrifugation 
after  one  and  one-quarter  hours  of  incubation  give  the  same  number  of 
ultramicrobes,  about  90,000,000.    The  inoculated  organisms  have  therefore 
increased  from  5  to  90  millions;  the  increase  being  in  a  proportion  of  about 


60  THE  BACTERIOPHAGE 

1 : 18.  And  this  increase  has  taken  place  in  apparently  a  very  abrupt  man- 
ner, only  to  be  explained  as  a  result  of  the  liberation  of  actual  colonies 
containing  an  average  of  about  18  germs.  We  will  see  by  ultramicro- 
scopic  examination  that  the  lysis  of  a  parasitized  bacterium  takes  place 
brusquely,  by  bursting. 

Tube  2.    Control.    Suspension  of  V '.  cholerae  plus  the  bacteriophage. 

Counts  made  immediately  after  inoculation  of  the  bacteriophage  give: 
for  the  centrifuged  material  201,  for  the  non-centrifuged,  211  plaques. 

After  thirty  minutes  the  counts  are:  for  the  centrifuged,  210;  for  the  non- 
centrifuged,  216. 

After  one  hour  the  counts  are:  for  the  centrifuged,  203;  for  the  non- 
centrifuged,  199. 

After  one  and  one-half  hours  the  non-centrifuged  suspension  gives  207. 

Tube  3.  Control.  Sterile  bouillon  plus  the  bacteriophage.  The 
counts  immediately  after  the  inoculation  are:  for  the  centrifuged,  206; 
for  the  non-centrifuged,  210. 

After  thirty  minutes  the  corresponding  counts  are:  201  and  211. 

After  one  hour,  the  counts  are:  203  and  206. 

After  one  and  one-half  hours  the  non-centrifuged  medium  contains  198. 

As  is  evident,  in  the  absence  of  bacteria  capable  of  being  attacked,  noth- 
ing happens.  The  ultramicrobes  remain  inert  in  the  liquid. 

The  nature  of  the  multiplication  taking  place  in  the  presence 
of  the  Shiga  bacillus  does  not  permit  of  any  doubt  on  the  follow- 
ing points. 

1.  After  a  contact  of  thirty  minutes  at  37°C.  the  ultramicrobes 
have  almost  entirely  disappeared  from  the  fluid;  they  are  fixed 
by  the  bacteria.    After  one  hour  the  situation  is  essentially  the 
same. 

2.  After  one  and  one-half  hours  there  is  an  abrupt  increase  in 
the  number  of  the  bacteriophagous  ultramicrobes. 

3.  The  fixation  is  elective;  it  does  not  occur  with  V.  cholerae, 
for  example,  for  which  the  bacteriophage  in  question  is  without 
action. 

From  this  it  may  be  concluded  that  the  culture  of  the  ultrami- 
crobes takes  place  within  the  interior  of  the  bacillary  body,  and 
all  the  other  observed  facts  support  this  conception  based  upon 
experiment.  We  can  now  understand  the  cause  for  the  successive 
jumps  noted  in  the  culture  of  the  bacteriophage,  mentioned  pre- 
viously in  connection  with  the  multiplication  of  the  germs.  Each 
of  the  ultramicrobes  inoculated  penetrates  to  the  interior  of  a 
bacillus  and  there  multiplies  up  to  the  time  when  the  bacillary 


BACTERIOLYSIS  61 

body  bursts.  This  liberates  the  colony  of  ultramicrobes  which 
have  been  formed  in  the  bacterial  protoplasm.  A  confirmation 
of  this  fact  is  obtained  by  examination  of  the  phenomenon  under 
the  ultramicroscope,  and  by  a  study  of  the  temporarily  inhibitory 
action  of  an  anti-bacteriophagous  serum. 

It  has  been  shown  that  the  successive  increases  in  number  are 
separated  by  intervals  of  approximately  seventy-five  to  ninety 
minutes.  On  the  other  hand,  a  complementary  experiment, 
conducted  in  the  same  fashion,  but  centrifuging  the  suspension 
at  ten  minute  intervals  during  the  first  half  hour,  has  shown  that 
very  few  of  the  bacteriophagous  germs  are  fixed  during  the  first 
ten  minutes,  although  they  are  almost  all  fixed  after  twenty 
minutes.  The  union,  therefore,  requires  about  a  quarter  of  an 
hour. 

Given  the  rapidity  of  multiplication  of  the  ultramicrobe,  and 
the  time  consumed  in  effecting  each  successive  increment,  it  can 
readily  be  calculated  that  a  single  bacteriophage  within  a  bacterium 
produces  a  colony  varying  in  number  from  fifteen  to  twenty-five 
individuals;  and  it  does  this  within  the  space  of  an  hour  or  an 
hour  and  a  quarter. 

BACTERIOLYSIS   UNDER   THE   MICROSCOPE 

The  anti-Shiga  bacteriophage  is  always  taken  as  an  example 
in  considering  the  action  on  dysentery  bacilli. 

It  has  been  seen  that  when  the  inoculation  with  the  bacterio- 
phage is  massive  all  the  bacteria  are  attacked  at  the  beginning, 
in  other  words,  their  multiplication  is  abruptly  arrested.  After 
two  to  three  hours  the  medium  commences  to  clear  little  by  little 
and  becomes  completely  limpid  a  short  time  later.  If,  on  the 
contrary,  the  inoculation  is  minimal,  the  few  ultramicrobes  inocu- 
lated only  affect  an  equal  number  of  bacteria.  The  great  majority 
remain  unaffected  and  multiply  as  they  would  in  a  normal  medium. 
But  the  ultramicrobes  likewise  multiply,  following  a  progression 
more  rapid  than  that  pursued  by  the  bacteria,  so  that  within  a 
few  hours  their  number  becomes  equal  to,  or  greater  than,  that  of 
the  bacteria.  This  is  the  time  when  macroscopic  lysis  commences. 

1.  Let  us  consider  the  first  case,  that  of  the  massive  inocula- 
tion. If  we  take  from  time  to  time  a  drop  of  the  suspension 


62  THE   BACTERIOPHAGE 

up  to  the  point  when  lysis  is  complete,  spread  these  drops  on 
slides  and  stain,  either  with  the  Gram  stain,  with  carbol-thionin, 
or  by  the  Romanowsky-Giemsa  method  (all  staining  methods 
give  essentially  the  same  picture),  results  such  as  the  following 
are  secured. 

A  suspension  of  Shiga  bacilli,  250,000,000  per  cubic  centimeter,  is  inocu- 
lated with  0.1  cc.  of  a  culture  of  the  bacteriophage  and  incubated  at  37°C. 

After  fifteen  minutes  it  appears  as  a  culture  of  normal  bacilli. 

After  thirty  minutes  it  appears  essentially  the  same,  except  that  a  few 
of  the  bacilli  are  poorly  stained. 

After  forty-five  minutes  about  10  per  cent  of  the  organisms  stain  poorly. 

Between  one  and  two  hours,  the  number  of  bacilli  which  stain  badly 
continues  to  increase,  and  after  2  hours  only  a  rare  cell  can  be  found  which 
has  taken  the  stain  normally.  At  the  same  time,  amorphous  debris  and 
granulations,  derived  most  certainly  from  the  bacteria  already  lysed,  are 
seen.  Similar  material  is  seen  very  abundantly  in  old  normal  cultures  of 
the  Shiga  bacillus.  These  granulations  dissolve  more  slowly  than  the 
remaining  portions  of  the  bacterial  protoplasm.  Finally,  and  this  is  a  most 
important  point,  spherical  forms,  more  or  less  ellipsoidal,  of  variable  dimen- 
sion, always  rare,  measuring  4  to  7  by  3  to  5  n  may  be  detected.  We  will 
see  in  a  moment  to  what  they  are  due.  There  are  occasional  bacillary 
forms,  well-stained,  having  a  length  of  from  8  to  12  p. 

Between  the  second  and  third  hours  the  amorphous  debris  considerably 
augments  and  the  bacillary  forms  rapidly  disappear.  A  few  spherical 
forms  are  still  to  be  seen. 

After  four  hours  lysis  becomes  more  and  more  complete.  Only  a  single 
poorly  stained  bacillus  will  be  found  in  two  or  three  fields. 

Gradually  the  formless  debris  disappears,  and,  in  turn,  the  granules. 
After  thirty-six  hours  nothing  whatever  can  be  distinguished  in  stained 
preparations. 

With  the  ultramicroscope  at  no  time  can  there  be  seen  elements 
other  than  the  bacilli  (whose  number  gradually  diminish,  to 
disappear  entirely  in  about  two  hours)  and  the  extremely  fine 
granules.  It  can  hardly  be  said  that  the  latter  represent  formed 
elements.  At  the  beginning  the  bacilli  present  a  normal  appear- 
ance. After  forty-five  to  sixty  minutes  fine  granules  are  seen, 
ever  becoming  more  and  more  abundant,  within  the  interior  of 
the  bacterial  cells.  The  number  of  bacterial  cells  containing 
granules  also  rapidly  increases  with  a  corresponding  diminution 
in  the  number  of  normal  bacilli.  The  most  interesting  part  of 
the  observation8  is  that  within  one  and  one-quarter  to  one  and 

8  First  noted  by  P.  Jeantet. 


BACTERIOLYSIS  63 

one-half  hours  after  the  beginning  of  the  process  the  bacilli  begin 
to  swell,  and  the  spherical  bodies,  containing  a  variable  number 
of  granules  (averaging  from  15  to  20)  are,  in  comparison  with 
a  normal  bacillus,  from  3  to  5  p  in  diameter.  If  the  spherical 
bodies  are  observed  with  care  it  is  seen  that  after  a  variable 
length  of  time,  sometimes  amounting  to  only  about  ten  minutes, 
an  actual  bursting  takes  place,  consuming  but  a  fraction  of  a 
second.  Immediately  afterward,  in  the  place  of  the  spherical 
body  there  remains  a  slight  cloudy  floccule  that  slowly  dissolves, 
thus  liberating  the  fine  granules.  These  spherical  bodies  are 
particularly  abundant  at  the  time  when  the  lytic  process  is  at  its 
maximum  rate.  There  can  be  no  question  concerning  the  nature 
of  these  bodies;  they  are  bacilli  which,  operated  upon  by  a  force 
which  can  only  be  internal,  take  at  first  a  globoid  form  and  then 
rupture.  This  is  the  more  certain  since  at  times  one  can  witness 
the  rupture  of  swollen  bacilli,  even  before  they  have  assumed  a 
spherical  contour.  This  observation  provides  direct  proof  that 
the  ultramicrobe  develops  and  exerts  its  action  within  the  bac- 
terial cell.  Destruction  of  the  bacilli  would  be  an  entirely  dif- 
ferent process  if  the  dissolving  action  were  exerted  on  the  exterior. 
The  spherical  form  and  the  bursting  prove  beyond  possible  con- 
tradiction that  the  operating  force  is  internal. 

What  do  the  fine  granules  that  can  be  seen  under  the  ultrami- 
croscope  represent?  While  nothing  can  be  affirmed  with  abso- 
lute assurance  there  is  nothing  to  preclude  the  supposition  that 
they  represent  the  ultramicrobes,  basing  this  upon  the  compara- 
tive examination  of  cultures  in  which  the  number  of  ultramicrobes 
has  previously  been  counted  by  plating  upon  agar.  By  such  a 
procedure  it  is  found  that  in  taking  two  cultures  presenting  a 
great  difference  in  count,  a  parallelism  is  always  to  be  noted  be- 
tween the  counts  and  the  number  of  granules  observed. 

It  would  likewise  be  well  to  recall  what  we  have  already  seen 
with  reference  to  the  multiplication  of  the  germs,  that  this  mul- 
tiplication appears  to  take  place  in  successive  jumps  (which 
correspond  to  the  rupture  of  a  large  number  of  parasitized  bacilli) 
and  in  which  the  number  of  ultramicrobes  liberated  after  one 
and  one-quarter  to  one  and  one-half  hours  corresponds  to  about 
eighteen  germs  to  each  single  one  inoculated.  And  we  will  see 


64  THE   BACTERIOPHAGE 

that  the  number  of  granules  consequent  upon  the  rupture  of  a 
cell  amounts  to  between  15  and  25.  There  is,  therefore,  a  great 
probability  that  the  granules  are  actually  the  ultramicroscopic 
bacteriophagous  organisms. 

2.  We  may  consider  the  second  case,  that  of  a  minimal  inocula- 
tion. In  this  case  the  medium  becomes  more  and  more  turbid 
before  lysis  actually  commences. 

A  suspension  of  Shiga  bacilli,  containing  250,000,000  per  cubic  centimeter 
is  inoculated  with  0. 0001  cc.  of  a  culture  of  the  bacteriophage,  a  very  active 
strain  being  selected. 

After  30  minutes  the  medium  has  its  original  turbidity;  essentially  that 
of  a  normal  culture  of  the  Shiga  bacillus. 

After  one  hour  the  original  turbidity  is  still  maintained .  When  smeared 
and  stained  all  the  bacilli  are  of  normal  shape,  but  an  occasional  form 
stains  poorly. 

After  two  hours  the  culture  is  about  twice  as  turbid  as  at  first.  There 
is  amorphous  debris  in  the  bottom  of  the  tube.  All  of  the  bacilli  appear 
to  stain  as  normally.  Many  of  the  bacilli  (about  two  in  every  three)  are 
about  four  times  the  normal  length,  that  is,  of  the  bacilli  used  to  seed  the 
culture,  and  there  are  all  intermediary  forms.  Oval  and  spherical  forms 
are  relatively  numerous,  but  they  are  always  fewer  than  would  be  expected 
from  a  comparative  ultramicroscopic  examination.  These  forms  are 
indeed  very  fragile  and  are  particularly  liable  to  destruction  during  fixation 
upon  the  slide  so  that  their  demonstration  in  stained  preparations  requires 
great  care. 

After  three  hours  the  suspension  is  slightly  cloudy.  The  bottom  of  the 
tube  is  covered  with  fine  debris  without  definite  form,  with,  from  place  to 
place,  great  amorphous  masses  and  numerous  granules  resembling  those 
encountered  in  very  old  cultures  of  normally  grown  Shiga  bacilli.  Only 
a  single  spherical  form  can  be  detected  in  a  ten-minute  search.  Each  field 
may  contain  a  dozen  large  bacilli,  well  stained. 

After  four  hours  the  turbidity  is  very  slight.  There  is  somewhat  less 
material  in  the  bottom  of  the  tube,  and  this  shows  only  a  single  poorly 
stained  bacillus  to  a  field. 

After  six  hours  the  medium  is  limpid.  There  is  still  less  deposit  in  the 
bottom  of  the  tube  and  it  is  with  difficulty  that  a  single  poorly  stained 
bacillus  may  be  found  in  searching  25  fields. 

After  eighteen  hours  nothing  at  all  can  be  seen  in  the  preparation. 

As  is  evident,  the  aspect  of  this  preparation  differs  but  little 
from  that  seen  in  the  former  case,  the  only  departure  being  that 
the  bacilli  which  have  grown  immediately  after  inoculation,  be- 
fore the  action  of  the  bacteriophage  becomes  operative,  present 
abnormally  large  forms. 


BACTERIOLYSIS  65 

A  comparable  ultramicroscopic  examination  in  the  two  cases 
shows  that  in  the  last,  where  the  inoculation  was  made  with  a 
bacteriophage  which  was  extremely  active,  at  the  time  when  lysis 
occurs  with  greatest  intensity,  that  is,  between  two  and  three 
hours  after  the  inoculation,  the  spherical  forms  were  present  in 
greatest  numbers.  There  were  as  many  as  two  to  three  to  a  field, 
and  their  rupture  was  readily  observed.  When  lysis  is  once 
terminated  the  most  careful  search  fails  to  reveal  such  forms. 

It  is  here  fitting  to  recall  an  observation  already  made  which 
should  be  noted  by  those  wishing  to  investigate  the  subject. 
When  a  simple  diastatic  action  is  operative  it  proceeds  with  uni- 
form rhythm  when  under  identical  conditions.  This  is  not  the 
case  here.  Up  to  the  present  time  more  than  a  hundred  different 
strains  of  the  anti-Shiga  bacteriophage  have  been  isolated  and  no 
two  of  them  have  been  found  to  conduct  themselves  in  an  exactly 
identical  manner.  The  final  result  is  always  as  has  been  indicated, 
the  phases  of  the  phenomenon  always  progress  in  the  same  order, 
but  the  time  of  the  reaction  will  vary.  With  one  strain  of  the 
bacteriophage  complete  lysis  is  obtained  in  three  hours,  with 
another,  only  after  twelve  hours.  The  phases  follow  each  other 
in  one  case  four  times  more  quickly  than  in  the  other. 

Another  point  which  should  be  remembered  is  that  all  that 
which  has  been  said  up  to  the  present  time  has  been  in  reference 
to  bacteriophagous  strains  which  were  extremely  active;  that  is 
to  say,  strains  capable  of  producing  complete  lysis. 

A  summary  of  the  foregoing  shows  that,  in  so  far  as  the  micro- 
scopic observations  are  concerned,  there  is  no  time  when  one  can 
distinguish  in  stained  preparations,  whatever  the  magnification, 
microorganisms  other  than  B.  dysenteriae  Shiga.  Aside  from  the 
bacilli  one  can  see  only  formless  cellular  debris  becoming  more 
and  more  abundant  with  the  more  complete  destruction  of  the 
bacteria,  the  debris  later  dissolving  gradually.  Ultramicroscopic 
examination  indicates  that  the  ultramicroscopic  bacteriophagous 
germs  multiply  within  the  interior  of  the  bacilli,  and  this  observa- 
tion is  corroborated  by  all  experiments.  Such  examination  also 
indicates  that  very  probably  the  bacteriophagous  elements  are 
represented  by  the  very  fine  granules  which  can  be  observed, 
first  in  the  interior  of  the  bacilli,  and  later  in  the  ambient  fluid. 


UNIVERSITY  OF  CALIFORNIA 
DEPARTMENT  OF  CIVIL  ENGINES 
BERKELEY.  CALIFORNIA 


CHAPTER  II 

THE  BACTEKIOPHAGE  AND  THE  BACTERIUM 

Virulence  of  the  Bacteriophage.  Measure  of  Virulence.  Resistance  of 
the  Bacterium.  Secondary  Cultures.  Instability  of  Mixed  Cultures. 
Characteristics  of  Mixed  Cultures.  Resistant  Bacteria.  Acquisition 
of  Resistance.  Production  of  Anti-lysins  by  the  Bacteria.  Multiple 
Cultures. 

VIRULENCE   OF    THE   BACTERIOPHAGE 

In  the  preceding  chapter  we  have  taken  note  of  the  complexity 
of  the  phenomenon  under  study;  a  complexity  resulting  from  the 
fact  that  there  are  simultaneously  three  elements  which  react, 
the  one  upon  the  others, — the  medium,  the  bacteriophage,  and 
the  bacterium.  Moreover,  up  to  the  present  we  have  considered 
only  the  simplest  case  that  may  be  presented,  a  bacteriophage  of 
maximum  virulence  before  which  the  bacteria  always  succumb. 
Often,  the  issue  is  very  different,  and  for  two  reasons.  The 
activity  of  the  bacteriophage  is  not  fixed,  it  varies  along  a  scale 
extending  from  an  action  barely  capable  of  detection  to  a  lytic 
power  most  intense.  The  bacterium  on  its  part  is  not  passive; 
it  defends  itself. 

We  have  already  stated  that  the  activity  of  the  bacteriophage  is 
a  true  virulence,  in  the  exact  meaning  of  the  word,  "the  ability 
which  a  micro-organism  possesses  to  develop  within  the  body  of 
a  host  and  there  to  secrete  toxic  substances."  Just  as  for 
each  pathogenic  bacterial  type  there  is  a  scale  of  virulence,  so 
also  for  each  strain  of  bacteriophage  there  is  a  certain  virulence. 
It  is  possible  to  exalt  or  to  attenuate  the  virulence  of  a  given 
bacterium  and  the  same  can  be  accomplished  with  the  bacterio- 
phage. Finally,  just  as  higher  forms  when  parasitized  by  a 
bacterium  defend  themselves  and  are  capable  of  acquiring  an 
immunity  to  this  bacterium,  so  in  like  manner  the  bacterium 
attacked  by  a  bacteriophage  does  not  remain  passive,  but  strug- 
gles, and  may  either  be  destroyed  or  acquire  an  immunity.  All 
the  vicissitudes  of  a  conflict  between  an  animal  and  an  attacking 

66 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         67 

bacterium  are  duplicated  in  the  struggle  between  the  parasitic 
bacteriophage  and  the  attacked  bacterium.  The  resemblance  is 
complete.  It  is  only  a  matter  of  descending  a  degree  in  the  order 
of  size  in  the  beings  concerned. 

It  is  also  a  property  of  living  beings  to  never  be  the  same  at 
any  two  moments  of  their  existence.  If  the  phenomenon  of 
serial  transmissible  bacteriolysis  which  we  are  considering  were  of 
purely  diastatic  nature,  the  activities  as  they  unfolded  would 
follow  a  fixed  plan;  for  if  the  active  element  was  not  varied  in 
quantity  its  quality  would  in  all  cases  be  constant.  But  we  have 
seen  that  quite  the  contrary  is  the  case.  The  phenomenon  is 
independent  of  the  quantity  of  the  active  element  employed. 
The  dominating  feature  is  the  quality  of  this  element.  Such  are 
precisely  the  characteristics  of  vital  activities.  A  poison  acts  in 
accord  with  its  mass;  a  bacterium,  with  its  virulence. 

Experiment  has  already  shown  that  a  bacteriophage  but 
weakly  capable  of  attacking  an  organism  is  susceptible  to  increase 
in  potency  through  successive  passages  in  contact  with  the  bac- 
terium which  it  attacks.  In  order  to  recognize  the  differences 
presented  between  different  strains  of  the  bacteriophage  it  is 
preferable  to  work  with  strains  freshly  isolated  from  the  organism. 

Experiment  XV.  (A) .  Ten  cubic  centimeters  of  a  suspension  of  Shiga 
bacilli  are  inoculated  with  1  cc.  of  a  filtrate  made  directly  from  the  feces 
of  a  patient  with  dysentery.  The  suspension  is  held  at  37°C.  Counts  of 
the  ultramicrobes,  made  at  different  times  during  the  incubation,  give  the 
following  results  when  0.01  cc.  is  plated  on  agar. 

When  plated  immediately,  there  develop  16  plaques,  representing  1,600 
ultramicrobes  per  cubic  centimeter.  The  filtrate  from  the  feces  therefore 
contained  16,000  per  cubic  centimeter. 

After  one  and  one-quarter  hours,  the  count  is  40  plaques,  or  4,000  per  cc. 

After  two  and  one-half  hours,  a  1 :10  dilution  gives  42  plaques,  or  42,000 
per  cubic  centimeter. 

After  three  and  three-quarter  hours,  a  1 :100  dilution  gives  18,  or  180,000 
per  cubic  centimeter. 

After  five  hours,  a  1:1000  dilution  gives  4,  or  400,000  per  cubic  centi- 
meter. 

After  fourteen  hours,  the  lysis  is  not  complete,  the  medium  is  cloudy 
and  becomes  more  and  more  turbid,  so  that  after  forty-eight  hours  it  is  very 
turbid.  Here  there  is  an  abundant  culture,  but  lysis  is  never  complete. 
The  bacteria  have,  then,  acquired  a  certain  resistance  which  has  allowed 
them  to  reproduce  in  spite  of  the  presence  of  the  bacteriophage. 


68  THE  BACTERIOPHAGE 

A  result  of  this  kind  is  usual  when  the  filtrate  is  prepared  from  a  stool 
taken  shortly  before  the  manifestations  of  convalescence  appear. 

(B)  Ten  cubic  centimeters  of  the  Shiga  suspension  are  inoculated  with  1 
cc.  of  the  filtrate  prepared  from  the  feces  from  the  same  dysentery  patient, 
but  collected  24  hours  later,  the  patient  now  being  convalescent.  Counts 
of  this  mixture  give: 

When  plated  immediately,  no  plaques,  or  less  than  100  ultramicrobes 
per  cubic  centimeter.  Thus,  the  filtrate  contained  less  than  1,000  per  cc. 

After  one  and  one-quarter  hours  the  plating  shows  no  plaques. 

After  two  and  one-half  hours  there  are  9  plaques,  or  900  ultramicrobes 
per  cubic  centimeter. 

After  three  and  three-quarter  hours,  in  a  1:10  dilution,  there  are  27 
plaques,  or  27,000  per  cubic  centimeter. 

After  five  hours,  a  1:1000  dilution  shows  13  plaques,  representing 
1,300,000  per  cubic  centimeter. 

In  this  last  experiment  (B)  the  ultramicrobes  were  present  in 
the  filtrate  in  very  small  numbers,  certainly  less  than  1000  per 
cubic  centimeter,  that  is,  there  were  less  than  one-sixteenth  as 
many  as  in  the  filtrate  of  the  first  preparation  (A).  Nevertheless, 
the  suspension  was  completely  lysed  in  ten  hours  and  the  fluid 
remained  sterile  indefinitely. 

It  should  be  noted  that  the  multiplication  of  the  ultramicrobes 
was  much  more  rapid  in  the  second  experiment  than  in  the  first, 
and  that  this  corresponds  exactly  with  the  idea  of  a  greater  viru- 
lence. These  experiments  show  also  that  the  number  of  ultrami- 
crobes inoculated  is  without  effect  upon  the  intensity  of  the  phenom- 
enon, but  that  the  important  thing  is  the  quality  of  the  bacterio- 
phage, that  is,  its  virulence. 

It  is  significant  that  the  two  strains  of  bacteriophage  under 
discussion  were  derived  from  the  same  patient,  but  were  taken 
at  an  interval  of  twenty-four  hours.  It  is  the  same  bacteriophage 
whose  virulence  has  been  increased  in  vivo. 

It  would  be  possible  to  cite  a  great  number  of  experiments  of  the 
same  order.  On  each  page  of  this  text  facts  will  be  found  that 
show  that  the  essence  of  the  phenomenon  is  the  virulence  of  the 
bacteriophage,  a  virulence  extremely  variable,  exalted,  or  at- 
tenuated, or  indeed  absent  for  a  given  bacterium  according  to  the 
conditions  at  the  moment  obtaining.  This  extreme  variability 
observed  especially  in  vivo  is  due  to  a  variety  of  conditions.  It  is 
less  in  vitro,  where  we  are  able,  within  certain  limits,  to  secure  a 
relative  stability. 


THE   BACTERIOPHAGE   AND   THE   BACTERIUM  69 

EVALUATION   OF   THE   DEGREE   OF  VIRULENCE 

As  will  be  shown  in  Part  II  of  this  monograph,  it  is  necessary 
to  the  study  of  the  processes  of  immunity  associated  with  the 
presence  of  the  bacteriophage,  to  be  able  to  measure  as  exactly 
as  possible  the  degree  of  virulence  possessed  by  each  strain  of  the 
ultramicrobe.  The  intensity  of  the  action  upon  a  bacterial  sus- 
pension, or  on  a  culture,  in  a  liquid  medium  gives  an  indication 
of  the  virulence.  A  strain  of  maximum  activity  causes  complete 
lysis  in  a  relatively  short  period  of  time,  varying  between  three  and 
thirty  hours.  A  less  active  strain  causes  only  a  partial  lysis. 
This  method  of  evaluation  is,  however,  very  crude  and  subcultures 
upon  agar  provide  more  precise  determinations,  particularly 
when  dealing  with  strains  which  are  but  slightly  active. 

If  we  introduce  into  a  bacterial  suspension  a  drop  of  a  filtrate 
containing  a  bacteriophage  active  for  the  bacterium  in  the  sus- 
pension, and  if  we  plate  upon  agar  a  drop  of  this  material  after 
variable  periods  of  incubation,  it  is  possible  to  follow  the  multipli- 
cation of  the  ultramicrobes.  It  is  only  necessary  to  count  the 
isolated  colonies,  which  assume,  as  we  have  seen,  the  form  of 
circular  plaques.  If  working  with  two  or  more  strains  of  the 
bacteriophage,  it  is  thus  easy  to  follow  the  relative  rapidity  of 
their  multiplication,  and  by  the  same  fact,  to  measure  their 
respective  powers  of  growth  at  the  expense  of  the  bacteria  para- 
sitized, that  is  to  say,  their  virulence. 

The  extension  of  the  plaques  furnishes  a  second  measure  of  the 
rapidity  of  the  multiplication  of  the  ultramicrobes.  Each  plaque, 
representing  a  colony,  results  from  the  extension  into  the  culture 
of  the  descendants  of  a  single  ultramicrobial  element,  deposited 
during  the  plating,  at  the  expense  of  the  bacteria  in  its  environ- 
ment. The  more  rapid  the  multiplication  of  the  bacteriophage 
the  more  rapid  the  extension  of  the  plaque.  Thus,  the  diameter 
of  the  plaque  permits  a  valuation  of  the  degree  of  virulence  of  the 
bacteriophage  which  produces  it. 

Experiment  XVI.  The  relative  virulence  of  four  strains  of  an  anti- 
typhoid bacteriophage  taken  from  a  single  patient  convalescent  from 
typhoid  fever  (Jeanne  Del )  at  different  periods  during  this  convales- 
cence is  determined. 


70  THE  BACTERIOPHAGE 

A  suspension  of  B.  typhosus  containing  250,000,000  bacilli  per  cubic  centi- 
meter is  prepared  and  distributed  into  four  sterile  tubes,  10  cc.  to  the  tube. 
Each  of  these  tubes  is  then  inoculated  with  0.0001  cc.  of  a  filtrate;  the  first 
with  a  filtrate  prepared  on  the  first  day,  tube  2  with  that  prepared  on  the 
second  day,  etc.  After  shaking,  0. 1  cc.  is  taken  from  each  tube  and  plated, 
as  usual,  on  an  agar  plate,  taking  care  that  the  agar  layer  in  all  the  plates 
is  of  the  same  depth  simply  to  make  all  the  conditions  comparable.  After 
incubation  the  following  results  are  obtained : 

1.  The  filtrate  prepared  from  the  stool  taken  on  the  first  day  of  the 
appearance  of  the  bacteriophage  shows  16  very  small  plaques,  pin-point 
in  size. 

2.  The  filtrate  derived  from  the  stool  of  the  second  day  after  the 
appearance  of  the  bacteriophage  shows  31  plaques,   having  a  diameter 
of  less  than  1  mm.  each. 

3.  The  filtrate  made  from  the  stool  of  the  third  day  gives  52  plaques, 
with  diameters  of  about  2  mm. 

4.  The  filtrate  prepared  from  the  stool  of  the  fourteenth  day  shows 
42  plaques,  each  with  a  diameter  of  less  than  1  mm. 

Strain  3  is  by  far  the  most  virulent,  a  conclusion  that  is  supported  by 
the  fact  that  in  its  isolation  it  induced  a  total  lysis  of  the  bouillon  culture 
of  the  typhoid  bacillus.  Strains  2  and  4  are  much  less  virulent.  Only  by 
a  dozen  passages  was  it  possible  to  effect  an  enhancement  in  virulence 
sufficient  to  give  the  same  result.  And  at  that  time,  when  plated  on  agar 
the  plaques  had  a  diameter  of  2  mm. 

These  experiments  demonstrate  very  well  that  with  equal 
virulence  the  plaques  on  the  surface  of  a  medium  are  approxi- 
mately equal  in  size.  The  process  of  measuring  virulence  by 
counting  the  plaques  and  thus  determining  the  rate  of  multiplica- 
tion is  certainly  more  exact  than  is  observation  of  lysis  in  a  fluid 
medium.  It  is,  unfortunately,  too  complicated  to  be  applied  in 
routine  practice  when  a  large  number  of  strains  must  be  examined, 
as  is  the  case  when  working  with  patients. 

RESISTANCE   OF  THE  BACTERIUM 

In  the  first  of  the  two  experiments  just  cited  (Experiment  XV, 
A)  we  have  seen  that  the  bacterium  was  successful  in  developing 
in  spite  of  the  presence  of  the  bacteriophage.  The  virulence  of 
the  bacteriophage,  then,  although  constituting  a  most  important 
factor  in  the  phenomenon  is  not  the  only  consideration.  The 
bacterium  is  capable  of  resistance. 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         71 

Up  to  the  present  time  we  have  considered  only  the  case  where 
lysis  was  complete  and  permanent,  and  it  has  been  specifically 
stated  that  the  phenomenon  assumes  this  form  only  when  the 
bacteriophage  possesses  a  maximum  virulence  and  acts  upon  a 
limited  quantity  of  suspension — 10  to  20  cc.  In  spite  of  the  ful- 
fillment of  these  conditions  it  sometimes  happens  that  a  suspension 
which  has  been  lysed  in  a  normal  manner  with  a  perfectly  limpid 
appearance,  will  some  days  later  become  turbid.  Microscopic 
examination  shows  that  the  turbidity  is  due  to  multiplication  of 
the  bacteria,  and  tests  of  the  biologic  activity  prove  that  this  cul- 
ture is  composed  solely  of  bacteria  of  the  same  species  as  was  used 
in  preparing  the  suspension  upon  which  the  bacteriophage  was 
acting.  According  to  the  virulence  of  the  strain  of  the  bacteriophage 
being  tested  the  number  of  tubes  in  which  this  reaction  takes 
place,  that  is,  the  development  of  this  secondary  culture,1  is  more 
or  less  great. 

Inoculations  on  agar  or  in  bouillon  of  lysed  suspensions,  in  which 
secondary  cultures  later  develop,  remain  sterile  up  to  the  time 
that  the  secondary  culture  is  formed.  This  does  not  often  occur 
until  five  or  six  days  after  the  lysis,  sometimes  even  later. 

Experiment  XVII.  A  suspension  of  Shiga  bacilli,  containing  250,000,000 
bacilli  per  cubic  centimeter,  is  inoculated  with  0.001  cc.  of  a  culture  of  the 
bacteriophage.  Normal  lysis  takes  place  in  five  hours,  with  the  medium 
perfectly  limpid.  The  lysed  suspension  is  planted  on  agar  and  in  bouillon 
1,  2,  3,  4,  5,  6,  and  7  days  after  the  lysis  is  completed.  All  the  plantings 
remain  sterile.  On  the  eighth  day  the  lysed  suspension  is  slightly  clouded. 
On  the  ninth  day  a  drop  is  inoculated  into  broth  and  on  to  three  tubes  of 
agar.  Two  of  the  agar  tubes  remain  sterile,  the  third  shows  four  small 
colonies.  The  broth  tubes  give  a  culture  agglutinated  in  the  sediment. 

The  resistance  of  diverse  strains  of  a  single  bacterial  species 
is  not  constant.  Each  strain  appears,  on  leaving  the  organism, 
to  be  possessed  of  an  individuality  which  is  rapidly  effaced  by- 
successive  cultivations  upon  an  artificial  medium. 

1  In  order  to  facilitate  exposition  I  have  called  a  "secondary  culture" 
one  growing  again  in  a  lysed  suspension ;  a  "mixed  culture,"  the  inoculation 
into  a  nutritive  medium  of  a  "secondary  culture"  with  the  coexistence  in 
the  medium  of  bacteria  and  bacteriophage. 


72  THE   BACTERIOPHAGE 

In  the  following  experiment  the  most  powerful  strain  of  the 
bacteriophage  yet  isolated  is  made  to  act  upon  two  different  races 
of  the  Shiga  bacillus.  One  of  these  bacterial  strains  has  been  for 
a  long  time  under  artificial  cultivation,  being  used  by  the  Pasteur 
Institute  for  the  inoculation  of  horses  in  the  production  of  anti- 
dysentery  serum  (type  strain).  The  other  was  recently  isolated 
from  the  stool  of  a  patient  with  dysentery  (strain  Jerv.). 

Experiment  XVIII.  (A)  Twelve  tubes  of  the  suspension  of  the  type 
strain  of  the  Shiga  bacillus  are  each  inoculated  with  0.001  cc.  of  a  culture 
of  the  bacteriophage.  This  latter  has  been  carried  on  for  a  great  number 
of  generations  always  at  the  expense  of  a  single  bacillary  strain.  In  all 
twelve  tubes  lysis  is  perfect,  with  complete  clearing  in  four  hours.  After 
three  days  at  37°C.  one  of  the  tubes  is  slightly  cloudy,  the  others  are  clear, 
(Five  other  experiments,  each  consisting  of  12  tubes,  with  the  same  strain 
of  bacteriophage  and  the  same  bacillus  give  the  following  results: — tubes 
showing  secondary  cultures  in  each  set,  0,  2,  0,  3  and  1.  There  develop, 
then,  7  secondary  cultures  in  the  60  tubes,  or  12  per  cent.) 

(B)  Twelve  tubes  of  suspension  were  prepared  with  the  strain  Jerv.,  a 
strain  with  which  the  bacteriophage  in  question  had  never  been  in  contact. 
Each  of  these  tubes  is  inoculated  with  0.001  cc.  of  the  same  culture  of 
bacteriophage  as  that  used  in  the  preceding  experiment    (A).    Seven 
of  the  12  tubes  give  secondary  cultures.    The  results  from  five  other  experi- 
ments with  the  same  strains  are,  9,  5,  10,  5,  and  6  secondary  cultures,  or 
70  per  cent.    A  week  later  12  cultures  of  the  Jerv.  bacillus  are  inoculated 
from  one  of  the  previous  tubes  that  had  remained  clear.    From  these,  5 
secondary  cultures  are  secured. 

A  further  passage  made  after  another  week,  gives  4  secondary  cultures 
in  the  12  suspensions.  After  another  week,  a  fourth  passage,  still  taking 
the  bacteriophage  from  a  perfectly  limpid  culture,  yields  but  one  secondary 
•culture  among  the  twelve  inoculated. 

(C)  At  the  beginning  of  convalescence  in  the  dysentery  case    (Jerv.) 
a  bacteriophage  was  isolated  which  was  tested  in  the  same  manner  both 
on  the  type  Shiga  strain  and  on  the  Jerv.  strain.    This  last  was  derived 
from  the  patient  early  in  the  infection  at  a  time  when  the  intestinal  bac- 
teriophage had  manifested  no  activity  for  this  organism. 

With  the  bacteriophage  Jerv.  on  the  type  bacillus  4  secondary  cultures 
develop  among  the  12  suspensions  lysed. 

With  the  bacteriophage  Jerv.  on  the  bacillus  Jerv.,  there  are  no  sec- 
ondary cultures  among  the  12  tubes  lysed.  When  repeated  upon  an  addi- 
tional 12  suspensions  a  single  secondary  culture  develops. 

It  is  then  clear  that  the  anti-Shiga  bacteriophage  is  not  equally 
active  for  all  strains  of  B.  dysenteriae  Shiga.  This  fact  is  even 
more  in  evidence  with  other  bacterial  species,  for  example,  with 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         73 

B.  typhosus,  B.  coli,  B.  proteus,  and  B.  pestis.  Each  bacterial 
strain  possesses  an  individual  resistance,  particularly  when  freshly 
isolated,  which  renders  it  more  or  less  resistant  to  a  bacteriophage 
accustomed  to  an  in  vitro  existence.  Later  we  will  see  that  this 
resistance  increases  by  a  phenomenon  of  natural  selection. 

All  of  the  phenomena  in  which  the  bacteriophage  is  involved, 
whether  taking  place  in  vitro  or  in  vivo  (the  first  are  only  an  artifi- 
cial reproduction  of  the  last)  are  dominated  by  two  factors, — the 
virulence  of  the  bacteriophage  and  the  resistance  of  the  bacterium. 

THE  ORIGIN  OF  SECONDARY  CULTURES 

What  is  the  intimate  mechanism  of  the  process  that  results  in 
the  formation  of  secondary  cultures?  A  priori  two  hypotheses 
can  be  formulated.  Two  factors  are  present,  a  bacteriophage 
whose  virulence  may  be  attenuated,  and  a  bacterium  whose 
resistance  may  be  augmented.  Thus,  are  secondary  cultures  due 
to  a  weakening  of  the  activity  of  the  bacteriophage,  or,  do  there 
exist  in  the  bacterial  suspension  certain  individual  cells  which 
acquire  an  immunity  to  the  bacteriophage,  thus  leading  to  the 
development  of  a  resistant  race?  The  following  experiments 
clearly  settle  the  question  in  favor  of  the  last  hypothesis. 

In  the  chapter  treating  of  the  isolation  of  the  bacteriophage 
we  have  seen  that  in  the  large  majority  of  cases  the  strains  which 
are  freshly  isolated  are  of  too  low  activity  to  effect  a  complete 
lysis  of  a  bacterial  suspension;  cases  where  the  presence  of  the 
ultramicrobe  could  only  be  detected  by  the  presence  of  plaques 
upon  the  agar  slants.  These  same  strains  were  able  to  acquire, 
by  successive  passages,  a  very  high  activity,  a  potency  which 
enabled  them  to  bring  about  lysis  of  very  heavy  suspensions. 
This  method  of  serial  passages  of  the  bacteriophage,  in  which  it  is 
forced  to  develop  in  vitro  at  the  expense  of  a  given  bacterium, 
corresponds  exactly  with  the  method  of  Pasteur  for  effecting  an 
enhancement  in  virulence  of  a  bacterial  race  by  repeated  passage 
through  a  given  animal  species. 

This  single  experiment,  repeated  a  considerable  number  of 
times, — in  fact,  each  time  that  a  bacteriophage  of  low  virulence  is 
isolated  from  the  body — shows  that  secondary  cultures  are  not 
produced  by  a  simple  diminution  in  the  virulence  of  the  bacterio- 


74 


THE   BACTERIOPHAGE 


phage.  Indeed,  there  is,  on  the  contrary,  an  enhancement  with 
each  passage,  even  if  macroscopic  lysis  is  not  to  be  seen.  For  this 
the  following  experiment  offers  direct  proof: 

Experiment  XIX.  The  contents  of  a  tube  that  gave  a  secondary  culture 
(as  described  on  page  72)  is  filtered  through  infusorial  earth  and  a  bougie. 
Twelve  tubes  of  a  Shiga  suspension  are  inoculated,  each  receiving  0.001 
cc.  of  the  filtrate.  Perfect  lysis  is  seen  in  all  tubes,  and  in  all  but  one  the 
lysis  is  permanent.  This  single  tube  again  becomes  turbid  after  4  days. 

From  this  it  is  clear  that  the  bacteriophage  has  not  lost  in 
virulence,  and  that  secondary  cultures  can  not  be  ascribed  to  a 
change  in  that  direction.  The  bacteriophage  remains  virulent, 
coexisting  with  bacteria  which  have  become  resistant.  The 
secondary  cultures,  then,  are  the  result  of  an  adaptation  undergone 
by  the  bacterium  which  acquires  an  immunity  to  its  parasite. 

It  has  already  been  shown  that  the  number  of  ultramicrobes 
inoculated  is  without  influence  on  the  appearance  of  secondary 
cultures.  The  conflict  is  not  one  of  numbers;  it  is  rather  a  strug- 
gle in  which  the  significant  factors  are  virulence  on  one  side  and 
ability  to  resist  on  the  other. 

Experiment  XX.  A  suspension  of  B.  dysenteriae,  250,000,000  per  cubic 
centimeter,  is  distributed  into  6  tubes  and  these  are  inoculated  with  vari- 
able quantities  of  the  same  bacteriophage  culture.  The  following  results 
are  obtained: 


TUBE 

AMOUNT  OP 
BACTERIOPHAGE 
CULTURE 
INOCULATED 

RESULTS 

CC. 

1 

0.1 

Normal  lysis,  secondary  cultures 

2 

0.02 

Normal  lysis,  no  secondaiy  cultures 

3 

0.004 

Normal  lysis,  no  secondary  cultures 

4 

0.002 

Normal  lysis,  secondary  cultures 

5 

0.0002 

Normal  lysis,  no  secondary  cultures 

6 

0.00002 

Normal  lysis,  no  secondary  cultures 

The  tubes  yielding  secondary  cultures  are  distributed  at  ran- 
dom throughout  the  series,  showing  no  fixed  relationship  to  those 
tubes  in  which  lysis  was  permanent. 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM 


75 


Experiment  XXI.  This  experiment  shows  the  serial  activity  of  the  bac- 
teriophage  together  with  the  appearance  of  secondary  cultures.  Each 
tube  of  the  series  is  prepared  with  a  suspension  of  B.  dysenteriae,  250,000,000 
per  cubic  centimeter,  and  into  each  is  introduced  0.001  cc.  of  the  lysed 
suspension  of  the  preceding  tube.  Transfers  are  made  after  twenty-four 
hours,  that  is,  at  a  time  when  lysis  is  complete. 


A  FRESH  SUSPENSION  RECEIVED  THE 
MATERIAL  INDICATED 


July  8 
July  9 
July  10 

July  11 
July  12 
July  13 
July  14 

July  15 
July  16 
July  17 


0.001  cc.  of  bacteriophage  culture 
0.001  cc.  of  suspension  lysed  on  July  8 
0.001  cc.  of  suspension  lysed  on  July  9 

0.001  cc.  of  suspension  lysed  on  July  10 
0.001  cc.  of  suspension  lysed  on  July  11 
0.001  cc.  of  suspension  lysed  on  July  12 
0. 001  cc.  of  suspension  lysed  on  July  13 

0.001  cc.  of  suspension  lysed  on  July  14 
0. 001  cc.  of  suspension  lysed  on  July  15 
0. 001  cc.  of  suspension  lysed  on  July  16 


Permanent  lysis 
Permanent  lysis 
Secondary  cultures  in  3 

days 

Permanent  lysis 
Permanent  lysis 
Permanent  lysis 
Secondary  cultures  in  4 

days 

Permanent  lysis 
Permanent  lysis 
Permanent  lysis 


Certain  salts,  when  added  to  the  suspension  in  very  minute 
quantities,  0.1  mgm.  to  10  cc.  of  culture,  favor  the  development  of 
secondary  cultures.  The  salts  of  lead  (nitrate  and  acetate)  and 
of  silver  (nitrate  and  sulfate)  act  in  this  way.  The  soluble  phos- 
phates and  magnesium  sulfate  appear  to  be  without  action. 
With  a  single  strain  of  bacteriophage  and  a  given  strain  of  bacil- 
lus the  development  of  secondary  cultures  is,  in  general,  more 
frequent  when  the  suspension  is  prepared  from  agar  cultures 
several  days  old  than  when  made  from  fresh  cultures. 

At  first  thought  it  appears  strange  that  when  secondary  cultures 
develop  with  a  strain  of  bacteriophage  of  high  potency,  they  ap- 
pear in  some  tubes  and  not  in  others.  The  following  experiment 
offers  an  explanation  for  this. 

Experiment  XXII.  Two  flasks,  each  containing  200  cc.  of  a  B.  dysenter- 
iae suspension  (250,000,000  per  cubic  centimeter)  are  inoculated  with  0.04 
cc.  of  a  culture  of  the  bacteriophage  (the  same  strain  as  that  used  in  the 
preceding  experiments).  Immediately  after  inoculation  the  contents  of 
the  first  flask  is  distributed  into  20  tubes,  10  cc.  to  each.  In  all  of  these 
lysis  takes  place  normally,  being  permanent  in  19,  showing  a  secondary 


76  THE  BACTERIOPHAGE 

culture  in  1.  The  second  flask  is  portioned  out  the  next  day,  that  is,  after 
lysis  is  completed,  10  cc.  being  placed  in  each  of  20  tubes.  None  of  these 
become  turbid.  When  this  second  part  of  the  experiment  is  repeated,  18 
remain  clear,  and  2  tubes  yield  secondary  cultures. 

Each  flask  of  suspension  contained  50,000  million  bacilli,  and 
the  above  experiments  show  that  of  this  number  but  one  or  two 
were  capable  of  acquiring  an  immunity  to  the  very  active  bacterio- 
phage.  It  is  these  "immune"  bacilli  which  give  rise  to  organisms 
that  enjoy  the  same  degree  of  resistance. 

Secondary  cultures,  then,  have  their  origin  in  the  operation  of 
the  phenomenon  of  natural  selection,  whereby  some  bacilli  show 
a  greater  aptitude  than  others  to  the  acquisition  of  a  resistance  to 
the  bacteriophage. 

The  phenomenon  of  secondary  culture  formation  is  governed 
by  the  individual  properties  of  the  two  admixed  organisms, — 
bacterium  and  bacteriophage.  Against  a  single  strain  of  bac- 
terium the  less  virulent  the  bacteriophage  the  greater  will  be  the 
proportion  of  secondary  cultures,  or,  in  other  words,  the  greater 
is  the  number  of  bacilli  in  the  suspension  capable  of  acquiring  a 
resistance. 

Against  a  given  strain  of  bacteriophage  the  different  strains  of 
a  single  bacterial  species  are  not  endowed  with  an  equal  resistance. 
With  certain  strains  secondary  cultures  will  be  the  rule,  with 
others,  the  exception,  and  with  still  others,  they  will  never  occur. 

We  will  shortly  see  the  reasons  for  this  variation;  at  present  we 
may  say  that  the  degree  of  resistance  possessed  by  a  bacterium 
to  a  bacteriophage  is,  for  a  given  bacterial  species,  in  direct 
relation  to  the  degree  of  virulence  which  this  bacterial  strain 
possesses  for  the  higher  organism  which  it  is  capable  of  invading. 

INSTABILITY  OF  MIXED   CULTURES 

Mixed  cultures  result  from  a  state  of  equilibrium  between  the 
virulence  of  the  bacteriophage  and  the  resistance  of  the  bacterium. 
But  these  two  factors  are  by  nature  variable  and  vary  in  intensity 
from  one  time  to  another,  being  influenced  by  the  circumstances 
of  the  moment.  This  equilibrium  can  be  interrupted  experi- 
mentally in  either  direction,  so  as  to  favor  either  the  one  or  the 
other  of  the  factors. 


THE  BACTEBIOPHAGE  AND  THE  BACTERIUM         77 

For  example,  if  we  place  a  small  quantity  of  a  secondary  culture 
in  normal  saline,  or  preferably  in  30  per  cent  glycerine  bouillon, 
that  is  to  say,  in  a  medium  which  interferes  with  the  reproduction 
of  the  bacteria  and  which  exerts  no  destructive  action  on  the 
bacteriophage,  the  equilibrium  is  disturbed  in  favor  of  the 
bacteriophage. 

The  ultramicrobe  is  very  sensitive  to  the  action  of  acids,  and  if 
transfers  from  a  secondary  culture  are  made  upon  glucose  agar  it  is 
found  that  the  bacterium  reacts  upon  the  sugar,  acidifies  the 
medium,  and  breaks  the  equilibrium  in  favor  of  the  bacterium. 
The  bacteriophage,  not  being  able  to  exert  its  parasitizing  action, 
will  be  eliminated  after  a  few  transfers. 

Still  another  method  of  separation,  the  most  practical  of  all, 
consists  in  employing  the  method  used  by  Eliava  and  Pozerski 
(described  further  on)  for  obtaining  cultures  of  resistant  bacteria 
free  from  the  bacteriophagous  ultramicrobe.  It  is  only  necessary 
to  make  a  few  passages  on  agar  slants,  culturing  each  time  from 
the  extreme  upper  margin  of  the  agar  layer  where  the  medium  is 
somewhat  desiccated. 

An  ultrapure  bacterial  culture  can  also  be  obtained  by  the  use  of 
quinine,  since  this  substance  has  a  higher  antiseptic  activity  for 
the  bacteriophage  than  for  the  bacterium. 

We  have  seen  that  in  the  case  of  a  bacteriophage  but  slightly 
virulent  the  addition  of  the  filtrate  in  which  it  is  present  to  some 
bouillon  does  not  impair  the  development  of  the  inoculated 
bacteria;  it  is  only  by  spreading  cultures  on  agar  that  we  can 
detect  the  presence  of  the  bacteriophage  through  the  plaques  which 
develop  there.  To  increase  the  virulence  of  such  an  inactive  strain 
we  have  seen  that  it  is  necessary  to  make  several  successive  passages 
of  the  bacteriophage  along  with  the  bacterium.  Between  each 
passage  it  is  essential  either  to  filter  the  mixed  culture  through 
a  bougie  to  separate  bacteria  and  ultramicrobes  or  to  heat  the 
mixture  to  60°C.  to  destroy  the  bacteria,  while  leaving  the  bac- 
teriophage unharmed.  What  is  the  basis  for  this  technic?  The 
elimination  of  bacteria  which,  because  of  contact  with  the  bac- 
teriophage, are  defending  themselves  and  are  acquiring  a  certain 
degree  of  resistance,  a  resistance  which  permits  them  to  subsist  in 
spite  of  the  progressive  increase  in  virulence  of  the  bacteriophage. 


78  THE   BACTERIOPHAGE 

With  each  passage,  therefore,  we  force  a  bacteriophage  of  increas- 
ing virulence  to  act  upon  an  organism  of  normal  resistance,  that 
is,  upon  a  bacterial  suspension  lacking  resistance  at  the  moment 
when  it  is  placed  in  contact  with  the  bacteriophage.  Briefly 
stated,  this  technic  is  simply  a  method  of  opposing  the  develop- 
ment of  resistant  bacteria  by  natural  selection. 

THE  CHARACTERS  OF  MIXED  CULTURES 

Bacteriophage  of  low  virulence 

The  means  whereby  the  equilibrium  obtaining  in  mixed  cultures 
can  be  disturbed  have  been  mentioned.  It  is  of  interest  to  allow 
the  struggle  to  proceed  naturally  and  to  note  its  issue  in  a  medium 
where  each  organism  is  dependent  upon  its  own  resources,  that  is, 
to  permit  the  natural  selection  of  such  bacteria  as  are  most  apt 
in  the  struggle.  To  witness  this,  it  is  only  necessary  to  make  re- 
peated transfers  in  broth  without  intermediary  filtration  or  heating. 
In  cases  where  the  bacteriophage  is  of  low  virulence  in  vitro  the 
bacterium  usually  triumphs;  its  resistance  increases  little  by  little, 
the  more  vigorous  bacilli  survive  and  multiply,  and  the  point  is 
reached  where  the  ultramicrobes  no  longer  find  bacteria  suscep- 
tible to  invasion.  When  this  occurs  the  bacteriophage  ceases  to 
multiply  and  gradually  becomes  eliminated  from  the  culture, 
until  only  a  normal  culture  of  bacteria  remains. 

In  some  cases  the  state  of  equilibrium  is  more  stable  and  the 
mixed  cultures  are  able  to  continue  throughout  a  large  number  of 
passages. 

Often  these  mixed  cultures  show  cultural  abnormalities  and  a 
partial  lysis.  The  medium  may  become  turbid  only  to  become 
somewhat  cleared  later  and  finally  to  revert  to  a  turbid  condition. 

Experiment  XXIII.  Bouillon  is  inoculated  with  a  mixed  culture  (B. 
dysenteriae — bacteriophage)  taken  from  an  agar  slant  planted  thirteen 
months  previously  with  a  secondary  culture.  The  macroscopic  appear- 
ance passes  through  the  following  stages:  after  forty-eight  hours,  uniform 
turbidity;  after  five  days,  almost  completely  cleared;  after  thirteen  days, 
uniformly  turbid;  after  nineteen  days,  slightly  cloudy  with  some  sedimen- 
tation; after  one  month,  uniformly  turbid  with  sedimentation.  This  is 
the  final  appearance  and  at  this  time  the  bacterium  and  the  bacteriophage 
coexist  in  the  medium. 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM          79 

Transplants  into  bouillon  continue  to  give  mixed  cultures,  cloudy, 
with  less  marked  but  definite  changes  in  appearance.  These  alterations  in 
appearance  are  separated  by  intervals  of  only  a  few  hours. 

This  same  experiment  is  performed  with  another  strain  of  anti-dysen- 
tery bacteriophage,  the  inoculation  being  made  from  a  colony  on  a 
secondary  culture  two  months  old.  After  three  days  there  is  uniform 
turbidity.  In  five  days  the  medium  is  almost  limpid.  After  eleven  days 
it  is  very  cloudy  and  after  eighteen  days  it  is  clear  with  a  slight  sediment. 
All  subcultures  remain  sterile  and  the  medium  contains  a  very  active 
bacteriophage. 

In  the  mixed  cultures  with  changing  appearance  the  struggle 
between  the  bacteriophage  and  the  bacterium  inclines  first  in 
favor  of  one  contending  force  and  then  to  the  advantage  of  the 
other.  The  final  issue  is  at  times  in  favor  of  the  bacteriophage, 
at  times  in  favor  of  the  bacterium,  and  the  number  of  transfers 
necessary  to  bring  about  a  final  issue  is  extremely  variable. 

In  vitro,  the  struggle  generally  ends  with  the  bacterium  the 
victor.  In  Part  II  of  this  text  we  will  see  that  in  vivo,  with  mixed 
cultures  showing  fluctuations,  the  issue  of  the  struggle  is  deter- 
mined in  some  measure  by  the  superior  organism  (that  is,  the 
animal  body)  in  which  the  contending  forces  are  operating. 

Bacteriophage  of  very  high  virulence 

With  a  bacteriophage  of  very  high  virulence  secondary  cultures 
are  relatively  rare,  and  when  they  appear  they  offer  a  very  charac- 
teristic aspect,  at  least  in  so  far  as  B.  dysenteriae  is  concerned. 
The  medium  remains  perfectly  limpid,  the  bacterial  culture  ap- 
pears agglutinated,  multiplying  slowly  in  the  bottom  of  the  tube 
or  deposited  on  the  walls.  These  agglutinated  masses  may  attain 
a  size  as  large  as  the  head  of  a  pin  and  they  can  not  be  dissociated 
by  shaking.  With  other  bacteria  the  agglutination  is  less  marked. 

Subcultures  from  these  agglutinated  mixed  cultures  give, 
indefinitely  it  appears,  mixed  cultures  always  presenting  the  same 
appearance.2 

2  As  one  of  the  consequences,  very  important  from  the  practical 
point  of  view,  we  will  see  that  numerous  so-called  pure  bacterial  cultures 
are  to  be  found  in  most  laboratories  which  are  in  reality  mixed  cultures, 
contaminated  from  the  time  of  their  origin  with  a  bacteriophage. 


80  THE   BACTERIOPHAGE 

Two  different  strains  of  anti-dysentery  bacteriophage  have 
been  preserved  for  over  two  and  a  half  years  and  during  that  time 
they  have  undergone  more  than  100  successive  passages.  Never- 
theless, during  this  period,  repeated  tests  have  shown  in  these 
cultures  the  constant  coexistence  of  extremely  virulent  ultrami- 
crobes  and  of  bacteria  completely  refractory  to  several  very  viru- 
lent strains  of  the  anti-dysentery  bacteriophage. 

In  these  mixed  cultures  there  is  a  stable  equilibrium  between 
the  virulence  of  the  one  and  the  resistance  of  the  other,  and  in 
such  cultures  the  changes  in  appearance  previously  noted  will 
never  be  observed.  They  might,  indeed,  be  spoken  of  as  "sym- 
biotic cultures,"3  for  the  bacteriophage  can  not  be  cultivated  in 
series  unless  it  multiplies  and  it  can  not  multiply  unless  it  para- 
sitizes bacteria.  Moreover,  it  is  only  necessary  to  disturb  the 
equilibrium  in  favor  of  the  bacterium,  by  such  means  as  have 
been  mentioned,  to  cause  a  rapid  disappearance  of  the  ultrami- 
crobes,  rendering  them  henceforth  incapable  of  cultivation. 

All  of  the  bacteria  present  in  an  agglutinated  culture  are  to  be 
found  in  the  agglutinated  clumps,  none  are  free  in  the  medium. 
Subcultures  into  bouillon  from  the  clear  fluid  always  remain 
sterile,  no  colonies  develop  when  transferred  to  agar,  and  micro- 
scopic examination  fails  to  reveal  any  formed  elements.  All  the 
bacteria  there  present  have  assembled  in  the  agglutinate.  The 
clear  fluid  contains  only  the  extremely  virulent  ultramicrobes, 
as  may  be  proved  by  the  inoculation  of  a  bacterial  suspension 
which  quickly  becomes  lysed.  On  the  contrary,  as  we  have  seen 
above,  a  bouillon  subculture  made  from  the  agglutinate  always 
results  in  the  growth  of  a  mixed  culture. 

When  a  mixed,  agglutinated  culture  is  inoculated  into  a  pure 
culture  of  the  bacteriophage,  that  is,  into  a  suspension  previously 
inoculated  and  which  has  undergone  complete  lysis,  the  growth 
consists  of  an  agglutinated  culture,  just  as  though  the  inoculation 
had  been  made  into  fresh  sterile  bouillon. 

3  This  is  possible  if  we  interpret  symbiosis  in  a  broad  sense,  as  Noel 
Bernard  has.  The  definition  of  symbiosis  given  by  this  author  applies 
admirably  to  mixed  cultures:  "An  intermediary  condition  at  which  two 
antagonistic  organisms  arrive,  with  an  equilibrium  of  their  forces,  toler- 
ating each  other  in  a  prolonged  common  existence." 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         81 

If  some  of  the  agglutinate,  even  if  washed,  is  introduced  into  a 
suspension  of  B.  dysenteriae  lysis  takes  place  and  the  suspension 
becomes  perfectly  clear  within  five  to  six  hours.  Four  or  five 
days  later,  however,  the  agglutinated  masses  begin  to  appear  and 
gradually  increase  in  size.  The  ultramicrobes  contained  in  the 
agglutinate  used  as  inoculum  provoke  the  lysis  of  the  normal 
bacilli  of  the  suspension,  bacilli  which  are  non-resistant,  and  then 
later  the  resistant  agglutinated  bacilli  in  their  turn  reproduce  and 
the  result  is  that  which  would  have  been  secured  had  they  been 
inoculated  into  fresh  sterile  bouillon. 

All  stages  intermediary  between  these  two  extremes  may  be 
obtained;  cloudy  mixed  cultures  presenting  the  appearance  of  a 
normal  bacterial  culture  where  the  equilibrium  is  essentially 
unstable;  cultures  in  agglutinated  form  in  the  presence  of  a  per- 
fectly limpid  fluid,  representing  a  state  of  stable  equilibrium.  The 
medium  may  be  more  or  less  cloudy  with  the  bacterial  masses 
more  or  less  compact,  sometimes  having  but  little  density  forming 
a  coagulum.  The  type  of  the  mixed  culture  bears  a  relationship  to 
the  virulence  of  the  bacteriophage  and  to  the  resistance  of  the 
bacterium.  Hence,  the  appearance  of  the  mixed  culture  may  be 
as  variable  as  is  the  variability  in  the  properties  of  the  two  organ- 
isms which  are  present. 

Mixed  colonies  on  agar 

Instead  of  seeding  the  mixed  cultures  into  broth  they  may  be 
inoculated  on  to  a  solid  medium. 

Often  the  agar  will  remain  sterile,  indicating  that  the  equili- 
brium has  been  disturbed  in  favor  of  the  bacteriophage.  As  has 
been  said,  this  reaction  may  occur  with  inoculation  into  broth, 
but  it  is  more  frequent  when  agar  plantings  are  made.  It  appears 
that  the  bacteria  on  agar  are  more  readily  attacked  than  when  in 
a  fluid  medium.  For  this  there  may  be  several  reasons,  the  prin- 
cipal one  doubtless  being  the  proximity  of  the  bacteria.  The  first 
phase  of  the  struggle  is  certainly  associated  with  the  phenomenon 
of  chemotaxis.  In  a  fluid  medium  the  bacteria  in  suspension  are 
separated  by  considerable  spaces,  certainly  considerable  when 
compared  with  the  diameter  of  the  ultramicrobe.  Upon  solid 
media,  on  the  other  hand,  the  bacteria  actually  touch  each  other, 


S2  THE   BACTERIOPHAGE 

and  the  passage  of  the  parasitic  agent  from  one  bacterium  to 
another  is  readily  accomplished  since  a  strong  chemotactic  force 
is  not  required  to  bring  together  the  two  organisms  in  the  struggle. 
When  a  culture  develops  on  agar,  the  appearance  of  the  growth 
may  show  considerable  variation,  determined  by  the  relation 
between  the  resistance  of  the  bacterium  and  the  virulence  of  the 
bacteriophage.  These  two  factors  being  inherently  variables 
afford  an  infinite  number  of  possible  combinations,  resulting  in  an 
infinite  variation  in  the  possible  appearances  on  agar.  At  first 
sight,  one  of  two  principal  distinguishing  aspects  may  be  present: 

1.  A  smooth  layer  of  culture,  always  located  in  the  upper  portion 
of  the  agar  slant  where  the  medium  is  less  thick  (although,  it  is 
needless  to  say,  the  mixed  culture  may  be  distributed  over  the 
entire  surface  of  the  slant) .     The  extent  of  this  covering  layer  is 
variable.     Sometimes  there  is  only  a  fringe  of  bacterial  growth 
at  the  extreme  upper  margin  of  the  medium,  the  remaining  portion 
being  sterile.    At  other  times  the  culture  layer  covers  one-tenth, 
one-fourth,  one-third,  one-half,  or  even  three-fourths  of  the  slant, 
the  portion  remaining  sterile  always  being  the  lower  section  of 
the  slant  where  the  agar  is  of  greatest  depth.     The  cause  of  this 
is  simple.    We  have  seen  that  the  colonies  of  the  bacteriophage 
appear  as  circular  plaques,  apparently  sterile,  and  that  they  are 
of  greatest  size  where  the  agar  is  deepest.     The  reason  for  this 
has  been  stated,  and  the  present  instance  is  but  an  application  of 
this  general  fact. 

Certainly  most  bacteriologists  will  recall  having  seen  cultures 
of  this  character  without  having  recognized  the  cause.  As  a 
matter  of  fact,  many  cultures  are  to  be  found  among  culture 
collections  which  are  in  reality  nothing  but  mixed  cultures.  In 
all  cases,  when  one  observes  abnormal  cultures,  presenting  the 
characteristics  which  have  been  described,  one  may  be  sure  that 
such  a  mixed  culture  is  present,  that  is,  the  culture  is  one  which 
is  infected  with  the  bacteriophage. 

2.  The  culture  may  consist  of  more  or  less  numerous  isolated 
colonies,  even  when  the  medium  has  been  abundantly  inoculated. 

These  isolated  colonies,  in  their  turn,  may  present  different 
appearances,  associated  always  with  the  degrees  of  resistance 
and  of  virulence  of  the  bacterium  and  the  bacteriophage  respec- 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         83 

tively.  Three  types  of  colony  may  develop,  each  presenting 
individual  characteristics. 

a.  The  colonies  may  be  those  of  normal  dysentery  bacilli.4 
These  are  encountered  especially  when  working  with  mixed 
cultures  derived  from  the  inoculation  of  a  suspension  with  a 
bacteriophage  of  low  virulence.  But  even  with  a  very  virulent 
bacteriophage  all  of  the  colonies  may  appear  quite  normal. 

6.  Rare  colonies,  formed  only  of  cocci,  and  cultivable  under 
this  form.  They  may  be  grown  in  bouillon,  where  an  abundant 
culture  of  homogeneous  turbidity  is  secured,  or  on  agar,  where 
the  colonies  appear  somewhat  different  macroscopically  from 
those  of  a  normal  bacillus,  being  more  convex  and  more  opaque. 
Subcultures  obtained  by  the  inoculation  of  these  colonies  are  not 
mixed  cultures;  they  contain  only  cocci,  no  ultramicrobes  being 
present.  The  coccoid  form  is  maintained  during  a  number  of 
generations  and  then  the  bacterium  gradually  reassumes  its 
normal  form. 

c.  The  colonies  may  be  mucoid,  refractile,  difficult  to  dissociate, 
and  of  very  diverse  size — from  the  limit  of  visibility  up  to  those 
with  a  diameter  of  about  one  millimeter.  These  colonies  are 
cultivable  on  agar  and  reproduce  colonies  of  the  same  form. 
They  are  mixed  colonies,  and  in  them  the  simultaneous  presence 
of  both  elements,  bacterium  and  bacteriophage,  can  always  be 
demonstrated. 

Even  when  abundantly  seeded  upon  agar  these  colonies  never 
give  a  smooth  layer  of  growth  but  always  isolated  colonies,  more 
or  less  abundant,  and  always  of  variable  size.  Among  the  bac- 
teria of  the  inoculum  but  few  are  able  to  form  colonies.  There  is 
always  a  state  of  unstable  equilibrium  between  the  two  elements 
present:  the  bacterium  with  its  resistance,  and  the  bacteriophage 
with  its  virulence.  The  bacterium  forms,  or  does  not  form,  a 
colony  according  to  the  accidental  predominance  of  one  or  the 
other  of  these  factors.  This  is  especially  to  be  observed  when 
agar  is  seeded  with  the  agglutinated  masses,  for  however  abundant 
may  have  been  the  planting  only  very  rare  isolated  colonies,  all 
of  the  mucous  type,  develop. 

4  B.  dysenteriae  Shiga  is  simply  taken  as  an  example;  all  other  bacteria 
give  mixed  cultures  and  mixed  colonies  showing  quite  similar  appearances. 


84  THE   BACTERIOPHAGE 

The  cultures  secured  by  the  inoculation  of  the  mucous  colonies  on  differ- 
ent media  show  the  following  reactions: 

In  agar  stabs:  small  lenticular  colonies  about  the  needle  track. 

In  gelatine :  as  in  agar,  the  resistant  bacteria  remain  alive  and  cultivable 
for  at  least  eleven  months.  In  the  case  of  the  Shiga  dysentery  organisms 
this  represents  a  viability  at  least  ten  times  as  great  as  that  of  the  normal 
bacillus. 

In  gelatine  stabs:  large  opaque  colonies  with  opaque  centers. 

On  glycerine  potato  (prepared  as  for  the  cultivation  of  B.  tuberculosis}'. 
very  rare  colonies  on  the  potato,  very  abundant  growth  in  the  fluid  at  the 
bottom  of  the  tube. 

In  milk:  not  coagulated  in  ten  days. 

In  litmus  milk:  turns  to  mauve  after  two  months. 

On  coagulated  serum:  no  growth. 

In  neutral  red:  no  change  in  two  months,  either  on  agar  or  in  bouillon. 

In  litmus  milk  (Petruschky) :  acid  after  ten  days  and  remains  acid. 

When  the  mucous  colonies  are  suspended  and  heated  to  60°C. 
they  are  not  cultivable,  for  then  the  culture  contains  only  the 
living  very  virulent  ultramicrobe  which  is  not  killed  until  a  tem- 
perature of  about  75°C.  is  reached.  Reinoculated  into  bouillon, 
the  refractile,  mucous,  mixed  colonies  yield  two  types  of  culture, 
(a)  mixed  cultures  showing  changes  in  turbidity,  and  (6)  aggluti- 
nated cultures,  which,  as  we  know,  always  depend  upon  the  degree 
of  virulence  of  the  bacteriophage  and  the  capacity  of  resistance  of 
the  bacterium,  factors  which  regulate  the  appearance  of  the 
culture. 

We  have  seen  that  if  an  agglutinate,  taken  from  a  mixed  culture 
in  stable  equilibrium,  is  introduced  into  a  suspension,  a  lysis  of  the 
suspension  is  followed  by  a  growth  of  the  agglutinate.  The  same 
thing  transpires  if  an  abundant  seeding  is  made  on  tubes  of  slant 
agar  having  a  growth  of  the  Shiga  bacillus.  First,  plaques  appear, 
and  then  after  three  or  four  days  a  mucous  colony  develops  in  the 
center  of  each  plaque.  In  both  instances  the  bacteriophage  acts 
upon  the  normal  non-resisting  bacteria  and  dissolves  them,  then 
the  refractory  bacilli  multiply  as  they  would  have  done  on  a 
sterile  agar  or  in  bouillon. 

THE  RESISTANT  BACTERIUM 

From  that  which  has  preceded  it  may  be  deduced  that  the 
acquisition  of  resistance  by  a  bacterium  is  reflected  in  a  marked 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         85 

change  in  morphology.  We  have  seen  that  certain  colonies  on 
agar  are  composed  of  bacteria  presenting  the  coccoid  form,  other 
colonies  presenting  the  refractile  appearance  and  a  mucous  con- 
sistency. 

Coccoid  form 

The  following  experiments  are  interesting  for  they  allow  of  the 
appearance  of  the  coccus  form  with  a  later  return  to  a  normal 
morphology — the  first  in  the  colony  itself  at  a  few  days  interval, 
the  second  in  the  course  of  successive  passages. 

Experiment  XXIV.  A  Petri  dish  is  heavily  inoculated  from  an  agar  cul- 
ture of  B.  dysenteriae  and  is  placed  in  the  incubator  at  37°C.  for  about  four 
hours.  A  drop  of  the  bacteriophage  culture  is  then  placed  in  the  centre  of  the 
plate.  The  strain  of  bacteriophage  should  be  one  of  average  activity,  that 
is,  one  capable  of  regularly  causing  complete  lysis  of  a  bacterial  suspension 
but  with  which  secondary  cultures  usually  develop.  (With  too  virulent 
a  strain  the  area  where  the  drop  was  placed  remains  sterile  indefinitely.) 
The  plate  is  returned  to  the  incubator.  After  eighteen  to  twenty-four 
hours  a  layer  of  culture  composed  of  normal  dysentery  bacilli  develops, 
showing  in  the  centre  a  spot  devoid  of  growth,  apparently  sterile.  After 
thirty-six  to  forty-eight  hours,  the  spot  becomes  covered  with  extremely 
fine  colonies,  which,  when  examined  microscopically  are  composed  of 
cocci  only.  These  cocci  are  of  different  sizes,  from  1  to  4  /z  in  diameter, 
arranged  in  irregular  forms, — in  diplo-  and  in  tetrad  groupings.  Two 
days  later  microscopic  examination  still  shows  cocci,  but  among  them  are 
bacillary  forms  in  great  number.  Subcultures  on  to  agar  always  give  iso- 
lated colonies,  each  colony  always  reproducing  with  the  same  appearance 
and  with  the  same  sequence  of  forms, — first  a  coccoid  culture,  then  a  mix- 
ture of  cocci  and  bacilli.  These  cultures  always  contain,  moreover,  bacter- 
iophagous  ultramicrobes. 

Eliava  and  Pozerski  have  indicated  a  method  for  obtaining  the 
coccoid,  resistant  bacteria  free  from  admixture  with  the  bacterio- 
phage.5 These  bacteria  perpetuate  themselves  under  this  form 
for  a  certain  number  of  generations.  Return  to  the  bacillary  form 
occurs  gradually  and  after  about  fifteen  transplants  the  culture 
acts  as  a  normal  dysentery  organism  sensitive  to  the  bacteriophage. 

8  The  method  permits  the  purification  of  a  mixed  culture  by  the  elimina- 
tion of  the  bacteriophage.  To  accomplish  this  it  is  only  necessary  to  make 
transfers  on  agar  with  the  mixed  culture,  taking  for  inoculum  in  each 
passage  material  from  the  top  margin  on  the  agar,  as  near  the  edge  as 
possible. 


86  THE   BACTERIOPHAGE 

To  a  suspension  of  bacteria  in  bouillon  i's  added  0. 01  cc.of  abacteriophage 
culture  and  a  drop  of  the  material  is  spread  on  an  agar  slant.  Frequently 
the  medium  remains  sterile,  as  has  been  shown  above.  Sometimes  a  slight 
fringe  of  culture  is  secured,  always  at  the  upper  margin  where  the  medium 
is  somewhat  dried  out.  Material  from  this  fringe  is  planted  on  a  tube  of 
sterile  agar  and  after  incubation  a  culture  layer  studded  with  plaques 
is  found.  A  third  transfer  is  made  from  the  upper  portion  of  the  tube,  and 
this  is  continued  until  an  apparently  normal  culture  is  secured.  That  is 
to  say,  until  the  growth  develops  without  plaques.  At  that  time,  the 
culture  consists  of  resistant  bacteria,  free  of  bacteriophagCj  and  appearing 
coccoid  in  morphology. 

In  such  cultures  resistance  is  maintained  during  a  certain  num- 
ber of  passages.  Then  it  gradually  decreases  and  it  is  observed 
that  this  resistance  is  associated  with  the  coccoid  form,  for  the 
lengthening  of  the  bacterial  elements  affords  an  indication  that 
resistance  to  the  bacteriophage  is  decreasing. 

The  coccoid  form  may  be  secured  in  another  manner.  Certain 
of  the  agar  tubes  seeded  with  a  secondary  culture  show  after  a 
very  long  time — one  or  two  months,  or  even  more — a  colony 
situated  near  the  top  of  the  agar.  This  colony  increases  in  size 
slowly  and  may  attain  a  diameter  greater  than  one  centimeter. 
It  is  formed  solely  of  cocci.  There  can  be  no  doubt  but  that  it 
consists  of  modified  bacteria  since  successive  subculturings  yield 
normal  bacillary  forms. 

Atypical  colonies  have  been  secured  with  B.  dysenteriae  (Shiga, 
Flexner,  and  Hiss),  with  B.  coli,  B.  typhosus,  and  the  paraty- 
phoids. 

As  is  seen,  the  coccoid  form  is  most  certainly  a  resistant  form 
of  the  bacterium,  and  the  return  to  bacillary  form  taking  place 
gradually,  affords  an  index  of  the  decrease  in  resistance. 

This  morphologic  transformation  is  accompanied  by  a  profound 
change  in  the  properties  of  the  bacterium.  Coccoid  cultures  are 
not  agglutinable  by  a  specific  serum.  This  is  also  true  of  secon- 
dary cultures  and  mixed  cultures  iD  general.  Restoration  of 
agglutinability  is  coincident  with  loss  in  resistance  and  with  the 
return  to  normal  morphology. 

In  this  connection  it  has  been  shown  that  strains  of  B.  typhosus, 
inagglutinable  when  derived  from  the  body,  are  at  the  same  time 
composed  of  bacilli  which  are  resistant  to  the  anti-typhoid  bac- 


THE  BACTEKIOPHAGE  AND  THE  BACTERIUM         87 

teriophage,  and  further,  that  when,  by  subculture  on  agar,  they 
lose  their  resistance  they  also  become  agglutinable.  Inagglu- 
tinability  appears  to  be  a  property  of  the  bacteria  which  resist 
the  bacteriophage. 

The  vitality  of  resistant  bacteria  is  much  greater  than  that  of 
normal  bacilli.  For  example,  with  the  Shiga  dysentery  organism 
whose  vitality  is  weak  (there  are  but  few  strains  cultivable  after 
one  month,  none  among  the  numerous  strains  with  which  I  have 
worked  have  remained  alive  without  subculturing  for  more  than 
two  months),  all  of  the  colonies  on  agar  of  the  resistant  Shiga 
substrain  are  still  cultivable  after  eighteen  months. 

The  virulence  of  bacteria  which  are  resistant  to  the  bacterio- 
phage is  likewise  considerably  greater  than  that  of  normal  bacilli. 

Whatever  may  be  the  nature  of  the  resistant  bacteria,  and 
whatever  may  be  their  form,  there  is  no  doubt  but  that  they  can 
return  to  the  normal  form  with  normal  properties.  They  then 
behave  as  the  bacteria  of  the  same  species  which  were  used  to 
prepare  the  initial  suspension  upon  which  the  bacteriophage 
acted.  In  a  word,  there  can  be  no  question  either  of  an  accidental 
contamination  or  that  they  are  visible  forms  of  the  bacteriophage. 

With  a  coccoid  culture  of  B.  dysenteriae  Shiga  the  following 
biologic  reactions  have  been  effected,  reactions  which  indicate 
that  these  cocci  conserve  the  general  properties  of  the  normal 
dysentery  bacillus: 

a.  The  injection  into  a  rabbit  of  such  cultures  causes  the  death 
of  the  animal  with  paralysis  and  intestinal  lesions  identical  with 
those  observed  in  animals  killed  by  the  inoculation  of  typical 
dysentery  organisms. 

b.  Rabbits  immunized  with   carefully  graded   doses  of  such 
cultures  are  protected  against  a  surely  fatal  dose  of  typical  dysen- 
tery organisms. 

c.  The  serum  of  rabbits  which  have  been  treated  with  injections 
of  coccoid  cultures  contain  an  amboceptor  which  will  fix  com- 
plement in  the  presence  of  normal  bacilli. 

Zoogleic  form 

Microscopic  examination  of  the  agglutinate  formed  in  liquid 
media  by  bacteria  which  are  endowed  with  a  high  resistance, 
and  also  of  colonies  which  are  mucoid  on  agar,  show  that  the 


88  THE   BACTERIOPHAGE 

bacteria  (their  morphology  will  be  discussed  shortly)  are  sur- 
rounded by  a  mucous  material.  They  are  actual  zoogleic  colonies. 

As  has  been  said  above,  there  can  be  no  doubt  as  to  the  nature 
of  these  bacteria,  since  all  biologic  reactions  show  that  they  react 
as  did  the  bacteria  of  the  same  species  which  were  used  to  prepare 
the  original  suspension.  Moreover,  in  every  instance,  they  revert 
to  normal  form.  It  is  only  necessary  to  eliminate  the  bacterio- 
phage,  the  cause  of  the  transformation. 

It  is  thus  very  obvious  that  the  resistance  of  the  bacterium  to 
the  action  of  the  bacteriophage  profoundly  modifies  its  form. 
Resistance  accompanies  a  transformation  into  the  coccus  form, 
and  when  the  bacterium,  having  to  defend  itself  against  an 
extremely  virulent  bacteriophage  increases  its  resistance,  there  is 
formed  a  mucous  capsule  which  certainly  functions  by  hindering 
the  penetration  of  the  ultramicrobes  into  the  bacterial  body. 
Encapsulated  bacteria  thus  enjoy  a  refractory  state. 

The  transformation  is  accompanied  by  modifications  in  the 
properties  of  the  bacterium;  increase  in  viability,  enhanced  viru- 
lence, and  inagglutinability  by  specific  sera. 

The  resistance  persists  only  as  long  as  the  bacterium  must 
needs  resist  the  action  of  the  bacteriophage.  In  the  absence  of 
ultramicrobes6  the  resistance  gradually  falls,  somewhat  more 
rapidly  when  it  was  not  very  marked.  In  extreme  cases  it  disap- 
pears after  about  thirty  transfers  on  agar.  This  represents  a 
very  considerable  number  of  generations. 

We  have  seen  that  it  is  experimentally  possible  to  render  a 
bacterium  resistant  to  the  action  of  the  bacteriophage  and  we 
have  indicated  different  methods  which  enable  us  to  reproduce 
this  phenomenon  at  will.  It  is  not  an  artificial  phenomenon  but 
is  a  reproduction  of  a  natural  process  which  takes  place  within  the 
organism. 

Each  strain  of  a  species  of  a  pathogenic  bacterium,  recently 
isolated  from  the  body  of  an  individual,  offers  a  different  resistance 
to  the  action  of  the  bacteriophage,  and  this  resistance,  as  we  know, 
may  extend  to  an  absolutely  refractory  state.  These  differences 
of  resistance  arise,  as  will  be  demonstrated  later,  in  hereditary 

6  Suitable  means  have  been  indicated  for  purifying  a  mixed  culture  by 
eliminating  the  bacteriophage. 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         89 

factors  which  originated  in  the  struggle  which  took  place  between 
the  ancestors  of  this  bacterium  and  the  bacteriophage  within  the 
body  of  the  infected  animal.  As  is  the  case  of  the  resistance 
acquired  experimentally,  this  naturally  acquired  resistance  disap- 
pears gradually  with  repeated  culture  on  laboratory  media.  Thus, 
different  strains  of  a  single  species  of  bacteria  tend  to  become, 
by  a  gradual  process,  uniform,  and  after  a  sufficient  number  of 
transplantations  all  are  equally  sensitive  to  the  action  of  the 
bacteriophage. 

It  is  worthy  of  note  that  the  degree  of  virulence  of  a  bacterium 
is  strictly  in  relation  with  its  degree  of  resistance  to  the  bacterio- 
phage. We  will  have  occasion  to  demonstrate  this  frequently 
when  we  come  to  consider  the  relation  between  the  bacteriophage 
and  the  infections  due  to  bacteria. 

MICROSCOPIC   OBSERVATIONS 

In  the  agglutinated  and  the  zoogleic  colonies  certain  atypical 
forms  are  encountered,  which  are  also  to  be  found,  although  less 
abundantly,  in  mixed  cultures  in  general. 

Microscopic  examination  of  preparations  stained  with  thionin 
or  by  the  Giemsa  method  shows  a  variety  of  forms  depending 
somewhat  upon  the  age  of  the  colonies.  Up  to  the  second  or  third 
day  the  pleomorphism  is  considerable.  Together  with  the  typical 
bacillary  forms  and  the  cocci  there  are  elongated  bacilli,  some  as 
long  as  fifteen  micra,  with  all  intermediary  lengths;  some  straight, 
others  curved  at  all  angles.  Some  are  clubbed,  yeast-like.  Large 
and  small  granules  are  present  together  with  the  de"bris  derived 
from  the  destruction  of  the  different  forms.  All  stages  inter- 
mediary between  intact  forms  and  amorphous  de*bris  are  repre- 
sented. What  is  the  exact  significance  of  these  various  forms? 
They  are  forms  of  involution  or  forms  assumed  by  the  bacterium 
in  the  development  of  resistance.  That  is  all  that  It  is  possible 
to  say  with  certainty.  However,  some  of  these  forms  give 
the  impression  that  the  organism  in  question  is  reproducing  by 
sporogony. 

In  old  colonies  but  very  few  of  the  bacillary  and  coccoid  forms 
are  to  be  seen. 


90  THE   BACTERIOPHAGE 

The  subcultures  on  agar  can  be  multiplied  but  the  appearance 
is  always  the  same,  and  no  matter  which  of  the  colonies  is  selected 
the  biologic  tests  mentioned  above  always  show  that  they  are 
related  to  the  original  bacterium.  Further,  the  possibility  of 
reversion  to  the  bacillary  form  in  pure  culture  when  grown  on  a 
glucose  agar  medium  demonstrates  conclusively  that  contamina- 
tion has  not  taken  place. 

How  may  these  facts  be  explained?  Early  in  the  consideration 
of  the  phenomenon  the  hypothesis  presented  itself  that  in  these 
secondary  cultures  a  visible  form  of  the  bacteriophage  was  pres- 
ent. But  experiment  has  shown  that  this  interpretation  was 
false.  A  second  idea  has  developed  which  unfortunately  has  not 
been  completely  studied  for  lack  of  time  (the  subject  of  the 
bacteriophage  is  so  far-reaching  that  it  has  seemed  more  essential 
to  utilize  the  available  time  on  experiments  dealing  with  the  func- 
tion of  the  bacteriophage  rather  than  with  those  of  the  question 
of  morphology)  but  is  presented  only  that  it  may  be  considered 
by  those  who  are  particularly  competent  to  engage  in  morphologic 
studies. 

The  necessity  of  bacterial  intervention  for  the  production  of 
asci  by  certain  fungi7  is  a  condition  clearly  recognized  in  mycology. 
This  same  situation  may  intervene  among  bacteria  which  are 
resistant  to  parasitism.8 

This  hypothesis  is  perhaps  the  less  improbable  since,  according 
to  Schaudinn,  sexuality  is  a  fundamental  characteristic  of  living 
matter.  It  is  observed  with  B.  butschlii  and  with  B.  sporonema 
(in  addition  to  the  usual  mode  of  reproduction  by  transverse 

7  For  example,  Ascobolus  furfuraceus  (Moliard,  1903),  a  fungus  of  the 
genus  Willia  (Sartory,  1902),  and  an  aspergillus  growing  on  the  banana 
(Sartory,  1920). 

8  The  following  suggests  that  under  the  influence  of  the  bacteriophage 
non-spore-forming  bacteria  may  give  rise  to  filtrable  forms.    I  have 
noted,  although  rarely,  that  a  filtrate  obtained  by  passing  a  secondary 
culture  through  a  Chamberland  bougie  (L2  and  even  Ls)  becomes  turbid 
after  some  days.    Each  time  that  this  has  been  noted  the  turbidity  has 
been  due  to  the  growth  of  a  resistant  bacterium  such  as  was  present  in  the 
secondary  culture  prior  to  the  filtration.    The  conditions  under  which 
this  phenomenon  occurs  have  not  been  ascertained,  thus  the  observation 
is  simply  mentioned  without  emphasis  being  placed  on  its  interpretation. 


THE  BACTEEIOPHAGE  AND  THE  BACTERIUM         91 

division)  where  there  is  noted  an  autogamous  reproduction,  which 
implies,  at  least,  a  rudimentary  sexuality.  In  autogamy  there 
are  necessarily  differentiated  elements  which  fuse.  According 
to  Schaudinn  the  loss  of  sexuality  in  bacteria  is  an  indication  of 
a  degenerative  change  resulting  from  the  adaptation  to  a  para- 
sitic existence.  In  accordance  with  the  hypothesis  suggested 
above,  there  would  be  a  return  to  sexual  reproduction  as  a  means 
of  defense,  under  the  influence  of  the  parasitism  to  which  the 
organisms  have  been  subjected.  Such  sexual  reproduction  would 
be,  then,  extremely  frequent  in  vivo,  as  frequent  indeed  as  a  strug- 
gle between  the  bacterium  and  a  bacteriophage  occurs  in  the 
body.  In  Part  II  of  this  monograph  it  will  appear  that  this 
struggle  is  continuous. 

THE  ACQUISITION   OF  RESISTANCE 

How  can  this  acquisition  of  immunity  by  a  bacterium  be 
explained?  Numerous  experiments  have  shown  that  if  a  certain 
quantity  of  a  slightly  active  culture  of  a  bacteriophage  is  introduced 
into  a  relatively  heavy  (1000  to  2000  million  per  cubic  centimeter) 
suspension  of  bacilli,  the  ultramicrobes,  readily  demonstrated  at 
first  by  the  presence  of  plaques  on  plantings  on  agar,  disappear 
from  the  medium  after  an  interval  of  time  varying  from  one 
hour  to  two  or  three  days,  and  that  they  can  not  later  be  dem- 
onstrated. Subcultures  give  normal  cultures  of  bacteria.  On 
the  other  hand,  we  have  seen  that  with  a  very  virulent  bacterio- 
phage the  ultramicrobes  disappear  from  the  fluid  between  ten  and 
twenty  minutes  after  introduction  into  a  suspension,  but  that  they 
reappear  in  about  twenty  times  as  great  a  number  in  from  one  to 
one  and  a  half  hours  later — they  have  multiplied  within  the 
interior  of  the  bacteria.  In  the  case  of  a  bacteriophage  of  low 
virulence  it  seems,  therefore,  that  penetration  of  the  bacteria 
takes  place  but  that  multiplication  can  not  be  effected.  The 
bacterium  resists  and  the  ultramicrobe  is  actually  destroyed  in 
vivo.  These  parasitized  bacteria  which  "recover"  acquire  by 
this  an  immunity.  We  will  see  later  that  they  are  even  capable 
of  producing  antilysins. 

Another  fact  has  been  sometimes  observed  which  shows  that 
certain  bacteria  are  able  to  become  "carriers."     As  has  been  said, 


92  THE   BACTERIOPHAGE 

heavy  suspensions  which  are  inoculated  with  a  filtrate  containing 
a  relatively  avirulent  bacteriophage  give  after  a  few  hours  abso- 
lutely normal  cultures  on  agar,  free  of  plaques.  If  serial  trans- 
plants are  made  of  these  cultures,  inoculations  made  in  such  a 
manner  as  to  yield  an  even  layer  of  growth,  it  sometimes  happens 
that  after  a  certain  number  of  transplants,  two  to  four,  a  very 
definite  plaque  appears,  which  is  indeed  a  colony  of  the  bacterio- 
phage. This  is  evidenced  by  the  fact  that  successive  passages 
from  this  plaque  yield  a  very  active  bacteriophage.  From  whence 
could  this  ultramicrobe  have  so  suddenly  come?  The  ultrami- 
crobe  had  remained  alive  within  a  bacillus,  and  at  a  given  moment, 
it  overcame  the  resistance  of  the  latter  and  multiplied.  Its  viru- 
lence being  increased,  the  young  germs  were  able  to  parasitize 
the  neighboring  bacilli  and  form  a  colony.  Any  other  explana- 
tion seems  impossible,  since,  immediately  after  the  inoculation  of 
the  bacteriophage,  seeding  upon  agar  shows  plaques  characteristic 
of  the  presence  of  virulent  bacteriophagous  germs,  then  these 
germs  completely  disappear,  the  bacteria,  however,  remaining 
sensitive  to  the  action  of  a  more  active  bacteriophage,  for  perfect 
lysis  is  secured  if  the  suspension  is  inoculated  with  a  trace  of  a 
very  active  culture  of  the  bacteriophage,  and  finally,  the  active 
bacteriophage  reappears  after  a  series  of  subcultures  on  agar  in 
the  course  of  which  all  the  bacillary  cultures  have  been  normal. 
This  germ  can  only  be  one  of  those  which  had  disappeared.  The 
fact,  demonstrated  by  experiment,  of  the  penetration  of  virulent 
ultramicrobes  into  the  bacteria,  warrants  us  in  thinking  that  this 
ultramicrobe  (but  slightly  virulent)  has  been  preserved  in  a  latent 
living  state  within  the  interior  of  the  bacterium.  At  a  given 
moment  the  resistance  of  the  bacterium  is  broken  down  and  in- 
fection results. 

PRODUCTION   OP  ANTILYSINS   BY   BACTERIA 

By  its  mere  presence  the  bacteriophage  is  not  able  to  dissolve 
the  bacterium.  In  the  following  chapter  we  will  see  that  it  acts 
through  the  secretion  of  lysins  which,  however,  can  be  isolated 
free  from  the  ultramicrobe. 

The  following  experiment  shows  that  the  resistance  of  the 
bacterium  to  the  action  of  the  bacteriophage,  which  amounts  to 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM          93 

an  actual  immunity  in  the  true  sense  of  the  word,  is  in  great  part 
due  to  the  fact  that  the  bacteria  secrete  antilysins  which  neutralize 
the  lysins. 

Experiment  XXV.  A  very  active  strain  of  bacteriophage,  active  for 
B.  dysenteriae,  is  diluted  in  sterile  bouillon,  0.05  cc.  to  10  cc.  of  the  medium, 
and  0.05  cc.  of  this  dilution  is  introduced  into  a  second  10  cc.  of  medium. 
In  addition,  a  very  concentrated  suspension  of  B.  dysenteriae  is  prepared, — 
a  suspension  representing  a  twenty-four-hour  slant  agar  culture  in  6  cc.  of 
sterile  bouillon.  This  suspension  is  inoculated  with  0.05  cc.  of  the  second 
dilution  of  the  bacteriophage  culture  and  incubation  at  37°C.  for  four  days 
follows.  At  the  end  of  this  time  it  is  evident  that  the  suspension  is  not 
lysed  (because  of  the  too  great  quantity  of  bacilli)  but  when  a  drop  is 
planted  on  agar  the  medium  remains  sterile.  Therefore  the  bacteriophage 
has  multiplied.  This  heavy  suspension  is  filtered  through  a  bougie. 

To  each  of  three  tubes  containing  10  cc.  of  a  weak  suspension  of  dysen- 
tery bacilli  is  added — to  the  first,  0.05  cc.  of  the  filtrate  from  the  heavy 
suspension,  to  the  second,  0.05  cc.  from  the  first  tube,  and  to  the  third, 
0. 05  cc.  from  the  second.  After  an  incubation  period  of  twenty-four  hours 
it  is  seen  that  there  is  no  lysis  in  tube  1,  but  lysis  is  complete  in  tube  2, 
while  the  suspension  in  tube  3  is  still  turbid  although  it  clears  after  forty- 
eight  hours. 

But  one  conclusion  is  possible.  Since  lysis  did  not  take  place 
in  the  first  tube,  in  spite  of  the  presence  of  a  large  number  of 
virulent  ultramicrobes,  and  since  it  was  produced  in  the  other 
two  which  received  infinitely  less,  there  must  have  been  in  the 
filtrate  some  substance  which  inhibited  the  lytic  action  of  the 
bacteriophage.  This  was  not  manifested  in  the  last  two  tubes 
because  of  the  dilution.  It  is  likewise  because  of  dilution  that 
the  inhibiting  substance  did  not  manifest  its  action  on  agar. 
It  has  already  been  noted,  in  the  course  of  the  earlier  experiments, 
that  lysis  was  more  often  perfect  if  a  suspension  was  inoculated 
with  a  minimal  amount  of  the  bacteriophage  than  if  the  inocula- 
tion was  massive.  The  cause  for  this  fact  is  now  clear. 

This  experiment  is  in  all  respects  identical  with  that  described 
in  a  preceding  paragraph,  except  that  there  it  was  accomplished 
by  introducing  a  small  number  of  ultramicrobes  of  low  virulence 
into  a  large  number  of  bacilli.  Under  these  circumstances  the 
bacteriophage  is  overcome  and  destroyed  by  the  bacterium. 
In  this  last  experiment,  on  the  contrary,  we  have  substituted  for 
the  small  number  of  germs  of  low  virulence,  ultramicrobes  of  high 


94  THE   BACTEBIOPHAGE 

virulence.  These  ultramicrobes  develop  slowly,  but  at  the  same 
time  the  very  numerous  bacteria  have  had  time  to  adapt  them- 
selves and  to  elaborate  a  defensive  mechanism  to  the  lysins  se- 
creted into  the  medium.  They  have  produced  an  antilysin  which 
paralyzes  the  action  of  the  bacteriophage.  Moreover,  it  has  been 
shown  that  these  antilysins  are  specific,  for  those  secreted  by  the 
dysentery  bacillus  are  without  inhibiting  action  on  the  lysis  of 
B.  pestis. 

The  bacteria,  in  general,  react  like  higher  organisms.  B.  tetani, 
for  example,  secretes  a  toxin,  and  an  animal  which  would  be  killed 
by  a  large  dose  of  this  toxin  reacts  to  a  small  dose  by  the  produc- 
tion of  an  antitoxin  which  will  neutralize  the  toxin.  The  bacterio- 
phage secretes  a  lysin  and  the  bacterium,  which  could  not  resist 
a  rapid  attack,  produces,  when  placed  in  favorable  conditions,  an 
antilysin  which  neutralizes  the  action  of  the  lysin. 

We  will  further  see  that  the  organism  reacts  to  the  injection  of 
the  bacteriophagous  lysins,  which  in  effect,  are  nothing  but  foreign 
bodies  of  the  same  nature  as  the  toxins,9  and,  indeed,  in  a  manner 
quite  analogous  to  that  of  the  bacteria,  namely,  by  the  production 
of  specific  antilysins. 

MULTIPLE   CULTURES 

It  remains  for  us  to  consider  multiple  cultures.  In  the  following 
chapter  we  will  see  that  the  virulence  of  a  strain  of  bacteriophage 
is  rarely  limited  to  a  single  bacterial  species.  What  forms  do 
secondary  cultures  take  when  a  bacteriophage  is  forced  to  act 
upon  a  suspension  comprising  different  bacterial  species?  We 
will  confine  ourselves  to  the  most  simple  case, — a  bacteriophage 
reacting  upon  two  species  of  bacteria.  Let  us  take  as  an  example 
a  strain  of  bacteriophage  very  virulent  for  B.  dysenteriae  Shiga 
and  but  slightly  virulent  for  B.  coli,  as  evidenced  in  the  following 
experiment. 

Experiment  XXVI.  Three  tubes  of  bouillon  receive  respectively  0.01, 
0.1,  and  1  cc.  of  a  known  anti-Shiga  bacteriophage.  The  three  tubes  are 
then  lightly  planted  with  B.  coli.  Normal  cultures  develop  in  the  three 
tubes.  Platings  on  agar  give  few  plaques.  Each  of  the  three  cultures  is 

9  The  organism  does  not  defend  itself  against  a  toxin  because  it  is  toxic 
but  because  it  acts  as  a  foreign  colloid  in  the  body. 


THE  BACTERIOPHAGE  AND  THE  BACTERIUM         95 

re-inoculated  into  fresh  bouillon.  Normal  B.  coli  cultures  develop. 
Transfers  to  agar  give  two  plaques  for  the  first  tube,  none  for  the  other 
two.  The  culture  yielding  the  two  plaques  is  again  re-inoculated.  A  nor- 
mal culture  develops.  The  bacteriophage  has  been  eliminated. 

This  strain  of  anti-Shiga  bacteriophage  possesses,  therefore, 
an  extremely  feeble  virulence  for  the  strain  of  B.  coli  under  test. 

Experiment  XXVII.  To  10  cc.  of  bouillon  is  added  1  drop  of  a  concen- 
trated suspension  of  Shiga  bacilli  (this  should  give  a  slight  turbidity  equal 
to  about  50,000,000  bacilli  per  cubic  centimeter)  and  1  drop  of  an  equally 
concentrated  suspension  of  B.  coli.  This  double  suspension  is  then  inocu- 
lated with  0.01  cc.  of  the  anti-Shiga  bacteriophage  used  in  the  above 
experiment. 

After  twenty-four  hours  there  is  a  slight  turbidity.  A  new  passage  into 
a  double  Shiga-colon  suspension  is  made.  Perfect  lysis  takes  place  after 
eleven  hours. 

The  lysed  suspension  is  then  introduced,  in  a  quantity  of  0.04  cc.,  into  a 
simple  suspension  of  B.  coli.  Lysis  is  complete  in  seven  hours. 

The  ultramicrobes  have  developed  at  the  expense  of  the  Shiga 
bacilli,  and  thus  being  maintained  in  the  medium  they  have 
gradually  acquired  a  virulence  for  B.  coli. 

In  the  intestinal  tract  a  bacteriophage  never  finds  itself  in  the 
presence  of  but  a  single  bacterial  species.  And  this  experiment 
permits  us  to  comprehend  the  process  of  acquisition  in  vivo  of 
virulence  by  a  bacteriophage  for  a  given  bacterium. 


CHAPTER  III 
VIRULENCE  OF  THE  BACTERIOPHAGE 

Multiple  Virulence. 
Persistence  of  Virulence. 
Bacterial  Species  Attacked. 

MULTIPLE   VIRULENCE 

A  strain  of  bacteriophage  freshly  derived  from  the  body  is 
rarely  active  against  a  single  bacterial  species.  Usually  it  at- 
tacks a  certain  number  of  species  at  this  time,  and  possesses  for 
each  of  these  a  variable  virulence. 

It  may  be  objected  that  this  by  no  means  opposes  the  concep- 
tion of  a  plurality  of  species  in  the  genus  Bacteriophage,  each 
species  acting  against  a  determined  bacterium.  This  question 
will  be  considered  in  detail  in  a  later  chapter.  For  the  time  being, 
however,  it  will  only  be  stated  that  all  experimental  work,  par- 
ticularly the  work  with  the  complement  fixation  reaction,  favors 
the  idea  of  unicity,  whatever  may  be  the  origin  of  the 
bacteriophage. 

There  is  but  one  bacteriophage,  but  as  isolated  from  the  or- 
ganism, there  is  an  infinite  number  of  strains,  each  possessing  the 
capacity  to  attack  diverse  bacteria.  Such  a  strain  may  possess, 
for  example,  a  very  high  virulence  for  B.  dysenteriae  Hiss,  an 
average  virulence  for  B.  coli,  a  low  virulence  for  the  Shiga  dys- 
entery strain,  a  very  weak  activity  for  B.  paratyphosus  B}  and 
none1  for  the  other  intestinal  organisms  tested. 

Another  strain  may  be  very  active  for  B.  coli  and  B.  typhosus, 
but  slightly  active  for  B.  dysenteriae  Hiss,  and  inactive  for  the 
other  bacteria  tested. 

1  It  is  evident  that  when  it  is  stated  that  a  given  strain  of  bacteriophage 
lacks  virulence  for  such  and  such  a  bacterium  it  must  be  understood  "a 
virulence  such  as  may  be  demonstrated  by  the  present  technic."  Early 
in  my  investigations  it  was  possible  to  detect  only  those  strains  possessing 
a  considerable  activity;  all  others  were  unnoticed.  The  technic  has  since 
been  improved,  but  at  the  present  time  it  is  most  certainly  not  perfect. 

96 


VIRULENCE   OF  THE   BACTERIOPHAGE  97 

It  is  manifestly  impossible  to  make  a  complete  analysis  of  a 
strain  of  bacteriophage,  for  to  do  so  would  necessitate  a  determina- 
tion of  its  activity  against  all  known,  and  even  against  unknown, 
bacterial  species.  With  a  given  filtrate  prepared  from  the  in- 
testinal contents,  we  can  affirm  that  a  bacteriophage  is  found 
there  at  the  moment  of  testing  because  of  its  activity  manifested 
toward  a  given  bacterium.  On  the  contrary,  it  is  not  possible 
to  conclude  that  none  is  present  simply  because  the  tests  were 
negative.  Investigating  the  activity  of  the  intestinal  bacterio- 
phage in  a  filtrate  from  the  feces  of  a  healthy  person  a  negative 
result  has  been  obtained  in  testing  against  the  intestinal  bacteria 
toward  which  it  was  expected  an  activity  would  be  evidenced. 
The  investigation  was  extended  to  the  most  varied  bacterial 
types,  and  finally  a  strain  of  bacteriophage  was  isolated  active 
against  an  organism  of  the  Salmonella  group  (bacillus  of  hog 
cholera).  This  strain  of  bacteriophage  was  cultivated  in  series 
and  an  active  bacteriophage  was  thus  secured. 

A  given  strain  of  bacteriophage  will  vary  from  time  to  time, 
either  in  the  body,  as  can  be  demonstrated  by  isolating  the  bac- 
teriophage each  day  from  the  feces  of  a  patient  during  the  course 
of  the  disease  and  during  convalescence,  or  it  may  vary  in  vitro, 
as  has  been  shown  in  the  preceding  chapter. 

All  combinations  of  virulence  are  possible,  both  as  to  quantity 
and  to  quality;  that  is  to  say,  in  the  extent  of  the  action  against 
varied  bacterial  species,  and  in  the  intensity  of  the  action  for 
each  of  the  bacteria  attacked.  It  can  be  readily  seen,  in  view  of 
the  infinite  number  of  combinations  possible,  that  two  strains 
of  bacteriophage  identical  in  all  respects  can  not  exist. 

PERSISTENCE  OF  VIRULENCE 

The  faculty  which  a  strain  of  bacteriophage  possesses  to  return 
to  parasitism  with  a  bacterium  persists  throughout  a  very  great 
number  of  passages  in  vitro  along  with  a  bacterium  of  another 
species.  For  example,  in  1916  a  bacteriophage  was  isolated  which 
was  extremely  active  for  B.  dysenteriae  Shiga,  of  but  average  viru- 
lence for  B.  coli,  and  but  slightly  active  for  B.  typhosus  and  the 
paratyphoid  organisms.  This  strain,  which  has  been  used  in 
many  experiments,  was  subjected  during  the  years  1916,  1917, 


98  THE   BACTERIOPHAGE 

1918,  and  1919  to  a  large  number  of  passages,  somewhat  more 
than  1200,  always  with  the  dysentery  bacillus.  Nevertheless, 
early  in  1920  experiment  showed  that  it  had  an  average  virulence 
for  B.  coli  and  a  very  low  activity  for  B.  typhosus. 

The  action  on  the  typhoid  bacillus  of  a  bacteriophage  which 
had  received  more  than  a  thousand  passages  with  B.  dysenteriae 
is  evidently  weak.  This  can  be  demonstrated  by  spreading  on 
agar,  a  procedure  which  permits  the  formation  of  characteristic 
plaques.  If  we  introduce  into  a  tube  of  bouillon  about  ten  drops 
of  an  anti-dysentery  bacteriophage  and  then  a  small  amount  of 
typhoid  culture,  we  secure,  after  incubation  for  eighteen  to  twenty- 
four  hours,  a  culture  of  B.  typhosus  which  appears  normal,  but 
if  a  drop  is  seeded  upon  agar  a  few  plaques  are  obtained. 

The  following  observation,  made  by  G.  Eliava,  is  of  the  same 
nature,  but  more  typical,  for  there  is  a  crossed  reaction  on  bac- 
teria but  remotely  related.  A  strain  of  bacteriophage  isolated 
from  the  pus  of  an  abscess  (we  will  see  that  under  certain  cir- 
cumstances the  intestinal  bacteriophage  may  pass  into  the  cir- 
culation) and  very  active  against  Staphylococcus  aureus,  appeared, 
even  after  a  series  of  more  than  100  passages  with  the  staphylo- 
coccus,  to  be  endowed  with  a  degree  of  activity  for  the  dysentery 
bacillus. 

Experiment  XXVIII.  Ten  cubic  centimeters  of  a  Shiga  suspension  is 
inoculated  with  0.25  cc.  of  a  filtered  anti-staphylococcus  bacteriophage. 
When  plated  immediately  on  agar  a  normal  culture  of  B.  dysenteriae 
develops.  After  incubation  at  37°C.  for  twenty-four  hours,  0.02  cc.  of  this 
suspension  plated  on  agar  gives  4  plaques.  This  virulence  for  B.  dysen- 
teriae was  increased  by  successive  transfers  in  association  with  this 
organism. 

Such  experiments  allow  us  to  penetrate  further  into  the  phe- 
nomenon of  virulence  of  the  bacteriophage. 

We  have  introduced  into  the  suspensions,  either  of  B.  typhosus 
or  of  B.  dysenteriae,  several  hundred  millions  of  ultramicrobes, 
all  virulent  for  the  Shiga  bacillus  in  the  first  case,  for  the  staphy- 
lococcus  in  the  second.  Each  plaque  on  the  agar  represents  a 
colony  derived  from  a  virulent  ultramicrobe;  virulent  in  one  case 
for  the  typhoid  bacillus,  in  the  other  for  B.  dysenteriae.  We  have 
also  seen  that  of  the  several  hundred  millions  of  ultramicrobes 


VIRULENCE   OF  THE   BACTERIOPHAGE  99 

only  a  few  were  endowed  with  a  latent  virulence  sufficient  to 
parasitize  the  new  bacterium  offered  them  and  to  multiply  at 
its  expense. 

The  persistence  of  latent  virulence  is  the  appanage  of  certain 
particularly  apt  individuals.  With  each  passage,  at  the  expense 
of  the  new  bacterium,  these  individuals  multiply,  their  virulence 
becomes  enhanced,  and  finally,  after  a  sufficient  number  of  pas- 
sages, a  complete  lysis  of  the  suspension  is  secured. 

With  these  facts  as  a  basis,  an  attempt  has  been  made  to  adapt 
to  the  dysentery  bacillus  a  strain  of  bacteriophage  active  for  the 
staphylococcus,  although  the  ultramicrobe  at  first  appeared  de- 
void of  all  action  on  the  dysentery  strain.  In  this  attempt,  ten 
drops  of  an  active  anti-staphylococcus  bacteriophage  were  inocu- 
lated into  a  double  suspension  containing  in  each  cubic  centimeter 
ten  million  staphylococci  and  ten  million  B.  dysenteriae.  After 
twelve  passages  (each  passage  being  separated  by  a  bougie  filtra- 
tion and  twelve  drops  of  the  filtrate  inoculated  into  a  fresh 
staphylococcus-dysentery  suspension)  the  bacteriophage  very 
actively  attacked  the  dysentery  bacillus.  This  property  de- 
veloped very  abruptly  during  the  eleventh  passage.  After  a 
series  of  twenty  passages  made  in  conjunction  with  Shiga  alone 
the  bacteriophage  did  not  possess  any  activity  for  the  staphylo- 
coccus, and  it  has  been  impossible  to  cause  it  to  reassume  such 
activity. 

Up  to  the  present  time  it  has  been  impossible  to  accomplish 
the  inverse  experiment,  that  is,  to  cause  a  bacteriophage  active 
for  the  Shiga  bacillus  to  acquire  a  virulence  for  the  staphylococcus. 

This  inequal  persistence  of  latent  virulence  of  the  bacteriophage 
against  diverse  species  of  bacteria  may  be  explained.  The  in- 
testinal bacteriophage  maintains  itself  in  the  intestinal  tract  at 
the  expense  of  the  different  intestinal  bacteria.  The  bacterio- 
phage is  therefore,  in  reality  a  normal  parasite  of  the  colon- 
typhoid-dysentery  group  and  an  accidental  parasite  of  the  other 
bacteria  against  which  it  acquires  virulence  in  the  same  environ- 
ment, as  a  result  of  conditions  at  present  undetermined.  It  can 
be  understood  then,  that  a  bacteriophage  active,  when  derived 
from  the  body,  for  an  organism  rather  remote  from  the  colon- 
typhoid-dysentery  group,  for  example,  a  staphylococcus,  retains 


100  THE  BACTERIOPHAGE 

for  a  very  long  time,  probably  indefinitely,  the  capacity  to  attack 
a  bacterium  of  the  enteric  group,  and  this  in  spite  of  very  many 
passages  in  conjunction  with  the  bacterium  accidentally  attacked. 
On  the  contrary,  a  bacteriophage  possessing,  when  derived  from 
the  body,  a  virulence  for  a  bacterium  accidentally  attacked, 
loses  more  or  less  rapidly  this  virulence  if  it  is  maintained  in 
vitro  with  a  bacterium  normally  attacked,  that  is  to  say,  at  the 
expense  of  an  organism  of  the  colon-typhoid-paratyphoid-dys- 
entery group. 

It  may  be  objected  that  all  these  facts  may  be  interpreted,  not 
as  a  persistence  of  a  latent  virulence  but  as  a  persistence,  through 
the  course  of  successive  passages,  of  several  species  of  the  bac- 
teriophage. In  other  words,  there  has  been  a  "contamination" 
of  the  anti-dysentery  bacteriophage  by  an  anti-typhoid  bacterio- 
phagous  ultramicrobe  in  the  first  case  cited,  and  a  "  contamina- 
tion" of  the  anti-staphylococcus  bacteriophage  by  an  anti-dys- 
entery bacteriophage  in  the  second. 

Both  experimentation  and  mathematical  reasoning  show  that 
such  an  interpretation  is  false. 

In  the  first  example  cited,  it  has  been  shown  that  the  number 
of  ultramicrobes  virulent  for  B.  typhosus  did  not  vary  during  the 
course  of  the  passages.  The  number  of  plaques  obtained  on  agar 
by  inoculation  of  a  suspension  of  typhoid  bacilli  inoculated  with 
the  anti-dysentery  bacteriophage  is  essentially  the  same,  although 
the  bacteriophage  has  been  subjected  to  fifty,  one  hundred,  five 
hundred,  or  a  thousand  passages  at  the  expense  of  B.  dysenteriae. 
If  the  ultramicrobes  capable  of  attacking  the  typhoid  bacillus 
were  an  "impurity"  their  number  should  diminish  gradually  in 
the  course  of  the  transfers,  each  passage  being  a  dilution,  and 
they  should  quickly  disappear. 

If  one  calculates  the  extent  of  the  dilution,  after  a  thousand 
passages,  of  the  filtrate  which  served  as  the  original  inoculum 
for  the  bacteriophage  in  question,  a  figure  of  such  magnitude  is 
obtained  that  a  persistence  of  anti-typhoid  bacteriophagous  germs, 
throughout  the  series  of  successive  dilutions,  is  mathematically 
impossible.  One  can  calculate  readily  up  to  the  thousandth 
passage  (each  passage  consisting  of  the  inoculation  of  0.001  cc. 
of  filtrate  into  ten  cc.  of  bouillon).  The  value  of  the  dilution  in 


VIRULENCE   OF  THE  BACTEBIOPHAGE  101 

the  thousandth  tube  of  the  series  is  represented,  in  cubic  kilo- 
meters, by  the  figure  103982.  To  appreciate  this  incommensurable 
figure,  it  is  sufficient  to  say  that  with  only  the  twenty-second 
passage,  one  drop  of  the  original  filtrate  taken  from  the  feces  has 
been  diluted  in  a  number  of  cubic  kilometers  of  liquid  expressed 
by  the  number  1070.  That  is  to  say,  by  a  number  of  which  the 
logarithm  has  for  a  characteristic  70;  which  would  be  a  cube  of 
liquid  of  such  size  that  it  would  require  a  billion  centuries  for 
a  ray  of  light  to  pass  through  from  edge  to  edge.2 

The  action  is  not,  therefore,  to  be  explained  as  a  persistence 
of  anti-typhoid  bacteriophagous  germs  through  a  series  of  suc- 
cessive cultures. 

According  to  the  conception  of  Maurice  Nicolle  a  bacterium 
may  be  considered  as  a  mosaic  of  properties.  Each  of  these 
properties:  resistance  to  heat,  vitality,  virulence  for  such  and 
such  an  animal,  etc.,  is  susceptible,  within  a  single  individual, 
of  continuous  variation.  Within  a  bacterial  culture,  and  at 
any  given  moment,  no  two  individual  bacteria  can  be  found 
possessing  exactly  the  same  properties.  This  conception,  demon- 
strated by  daily  experience,  applies  moreover  to  all  living  beings. 

Variation,  that  is,  the  property  of  adaptation,  is  an  attribute 
of  life  and  of  life  exclusively.  Like  all  living  beings,  the  bacterio- 
phage  adapts  itself  continually,  and  in  any  culture  the  ultrami- 
crobes  which  compose  it  do  not  all  possess  exactly  the  same  prop- 
erties. Some  are  susceptible  of  rapid  adaptation  toward  a  given 
bacterium,  others  toward  another  organism.  A  bacteriophagous 
ultramicrobe  is  a  mosaic  of  properties. 

2  It  is  evident  that  the  same  mathematical  reasoning  demonstrates  that 
the  bacteriophage  is  itself  a  living  being.  If  one  would  wish  to  explain 
lysis  as  due  to  the  presence  of  a  lytic  diastase  in  the  intestinal  contents 
(or,  what  actually  amounts  to  the  same  thing,  the  presence  of  a  co-ferment 
or  a  catalyzer  in  the  intestinal  tract  capable  of  activating  a  pro-diastase 
contained  in  the  bacterium),  the  diastase  or  the  catalyzer  or  the  co-ferment 
would  be  quickly  eliminated  by  the  dilution.  If  we  suppose  possible  the 
persistence  of  one  of  these  principles,  in  spite  of  the  dilution  which  approxi- 
mates infinity,  and  its  presence  at  each  point  in  an  incommensurable 
amount  in  the  liquid,  we  are  endowing  this  principle  with  the  metaphysics 
of  ubiquity.  Any  conception  of  transmissible  serial  bacteriolysis  which 
does  not  admit  as  the  origin  of  the  phenomenon  an  autonomous  living 
being,  ends  in  a  mathematical  absurdity. 


102 


THE   BACTERIOPHAGE 


When  a  bacteriophage  is  virulent,  as  it  comes  from  the  organ- 
ism, for  several  bacteria  at  the  same  time,  as  is  the  usual  case,  it 
is  apparent  that  the  virulence  present  for  each  of  these  bacteria 
is  subject  to  variations  with  time.  This  is  true,  however,  the 
virus  may  be  preserved,  whether  it  is  kept,  sealed  in  tubes,  in  the 
original  intestinal  contents  or  whether  it  is  preserved  in  the  form 
of  filtrates  prepared  from  the  fecal  material. 

When  kept  in  vitro  certain  strains  of  bacteriophage  lose  their 
virulence  for  a  bacterium,  toward  which  they  were  active  when 
derived  from  the  body,  much  more  rapidly  than  do  others. 

Experiment  XXIX.    Typhoid   patient   Mor Examination 

of  the  stool  was  made  at  the  beginning  of  convalescence.  On  August 
20th,  1918,  the  stool  was  treated  according  to  the  method  described  for 
securing  the  bacteriophage.  The  filtrate  is  distributed  in  0.5  cc.  amounts 
in  suspensions  of  the  following  bacteria : — B.  dysenteriae  Shiga,  B.  typhosus, 
B.  paratyphosus  A,  B.  paratyphosus  B,  and  B.  coli.  After  24  hours  of 
incubation  these  suspensions  were  planted  on  agar  with  the  following 
results : 

B.  dysenteriae  Shiga Sterile 

B.  typhosus Sterile 

B.  paratyphosus  A Numerous  plaques 

B.  paratyphosus  B Numerous  plaques 

B.  coli Sterile 

Specimens  of  the  feces  and  of  the  filtrate  were  preserved  in  sealed  tubes. 
On  January  22nd,  1919,  that  is,  after  5  months,  these  materials  were  exam- 
ined again: 


SUSPENSION 

RES 

ULT 

Freshly  prepared  filtrate 

Original  filtrate 

B.  dysenteriae  Shiga.  

Sterile 

Sterile 

B.  typhosus 

Normal  culture 

Normal  culture 

B  .  paratyphosus  A  

Normal  culture 

Normal  culture 

B.  paratyphosus  B 

Numerous  plaques 

Numerous  plaques 

B.  coli 

Numerous  plaques 

Numerous  plaques 

In  this  material  the  virulence  of  the  bacteriophage  for  B.  dys- 
enteriae and  for  B.  paratyphosus  B  remained  unaltered  during 
the  five  months,  it  diminished  for  B.  coli,  and  disappeared  entirely 
for  B.  typhosus  and  B.  paratyphosus  A. 


VIKULENCE   OF  THE   BACTERIOPHAGE  103 

It  should  be  noted  that  the  result  was  the  same  whether  the 
bacteriophage  was  preserved  directly  in  feces  or  in  the  filtrate, 
that  is,  in  bouillon.  Likewise,  it  is  significant  that  the  degree 
of  virulence  has  no  influence  on  the  preservation  or  the  disappear- 
ance of  the  virulence.  It  was  strong  for  B.  typhosus  and  became 
negative,  it  was  weak  for  B.  paratyphosus  B}  yet  this  remained 
intact. 

In  the  absence  of  passages,  simply  as  an  effect  of  old  age,  the 
virulence  of  the  bacteriophage  varies  then  with  time,  and  indeed 
in  a  different  manner  for  the  diverse  bacteria  attacked.  It  be- 
comes attenuated  more  quickly  for  some  than  for  others,  and  for 
this  no  general  rule  can  be  fixed.  We  have  seen  above,  with 
another  strain,  that  after  four  years  and  in  spite  of  passages  in 
contact  with  the  dysentery  bacillus,  the  virulence  for  B.  typhosus 
persisted.  The  last  experiment  cited  is  not  only  interesting  then, 
in  that  it  shows  an  attenuation  of  virulence  associated  with  the 
lapse  of  time,  but  also  in  that  it  gives  evidence  that  the  loss  does 
not  occur  in  equal  degrees  for  all  of  the  bacteria  attacked  by  one 
and  the  same  bacteriophage. 

BACTERIAL  SPECIES  ATTACKED 

Let  us  now  consider  the  different  bacteria  for  which  active 
strains  of  the  bacteriophage  have  up  to  the  present  been  isolated.3 
For  each  of  these  only  the  peculiarities  of  the  reaction  as  they  are 

3  In  certain  cases  the  bacteriophage  can  serve  for  the  identification  of 
bacteria,  as  the  agglutination  reaction  is  used.  In  order  to  apply  the  test 
a  bacteriophage  must  be  employed  which  has  been  subjected  to  numerous 
passages  at  the  expense  of  a  particular  bacterial  type  so  that  the  accessory 
virulences  may  be  attenuated  as  far  as  possible.  For  example,  all  strains 
of  bacteria  which  are  lysed  by  a  strain  of  bacteriophage  that  has  been 
cultivated  together  with  Shiga  bacilli,  are  certainly  B.  dysenteriae  Shiga. 
With  certain  species,  B.  pestis  for  example,  for  which  the  specificity  of  the 
bacteriophage  appears  high,  diagnosis  by  means  of  the  bacteriophage  is 
particularly  conclusive.  In  this  last  connection  it  may  be  said  that  since 
the  publication  of  the  French  edition  of  this  text,  a  bacteriophage  has  been 
encountered  active  for  B.  pestis  and  equally  active  for  the  bacillus  found 
in  pseudotuberculosis  of  the  guinea  pig.  Thus,  it  is  necessary  to  recognize 
the  lack  of  an  absolute  specificity,  limiting  somewhat  the  value  of  the 
reaction  as  applied  to  the  identification  of  bacterial  species. 


104  THE   BACTERIOPHAGE 

encountered  in  dealing  with  the  bacterium  in  question  will  be 
mentioned.  As  far  as  general  characteristics  are  concerned,  all 
are  similar,  that  is,  what  has  been  recorded  in  preceding  chapters 
regarding  the  method  of  isolation,  the  mode  of  action,  the  variable 
virulence,  the  enhancement  of  virulence  by  passage,  the  resistance 
of  the  bacteria,  and  secondary  cultures,  applies  to  all  strains  of 
bacteriophage  and  to  all  species  of  bacteria  attacked.  In  all 
of  the  experiments  mentioned  up  to  the  present  time  the  dysen- 
tery bacillus  has  been  taken  as  an  example,  but  it  has  been  shown 
that  all  such  experiments  may  be  repeated  with  identical  results 
with  any  strain  of  bacteriophage  active  for  a  definite  organism. 
B.  dysenteriae  presents  practical  advantages  in  experimentation. 
It  is  easy  to  isolate  a  very  active  bacteriophage  for  this  organism 
and  bacteriologists  can  readily  procure  strains  and  repeat  these 
experiments  without  conducting  a  long  series  of  preliminary 
investigations. 

B.  dysenteriae  Shiga 

For  this  organism  it  is  particularly  easy  to  isolate  a  very  active 
strain  of  the  bacteriophage.  The  bacteriophage  opposed  to  this 
bacillus  exists,  it  may  be  said  to  be  normally  present,  in  the  in- 
testinal tract  of  numerous  animals,  the  horse  and  domestic  fowls 
m  particular.  It  is  likewise  frequent  in  man  and  may  acquire 
a  high  virulence,  not  only  in  convalescence  from  an  attack  of 
dysentery,  but  in  recovery  from  a  variety  of  pathologic  conditions. 
Up  to  the  present  time  about  200  strains  have  been  isolated, 
without  finding  any  two  of  exactly  comparable  virulence  and  of 
equal  extent  in  their  action  on  the  related  bacteria  of  the  colon- 
typhoid-dysentery  group.  Among  these  200  strains,  one  only, 
and  that  of  a  moderate  activity  when  isolated,  has  failed  to  act 
upon  any  other  bacteria  of  the  group. 

A  strain  of  bacteriophage  active  for  B.  dysenteriae  Shiga  is 
usually  active  for  B.  coli  and  for  B.  dysenteriae  Flexner  and  Hiss. 
From  the  point  of  view  of  the  bacteriophage  the  Shiga  type  of 
dysentery  bacilli  represents  a  homogeneous  species,  a  bacterio- 
phage active  for  one  strain  being  equally  active  for  all  others. 
A  bacteriophage  very  active  for  one  strain  of  bacilli,  at  the  ex- 
pense of  which  it  has  passed  through  a  number  of  passages,  may 


VIRULENCE   OF  THE   BACTERIOPHAGE  105 

be  less  active  for  a  freshly  isolated  race,  but  after  four  or  five 
passages  in  contact  with  this  strain  a  virulence  is  acquired  equal 
to  that  possessed  for  the  first  one. 

We  have  seen  that  the  strains  of  Shiga  bacilli  which  resist  the 
action  of  the  bacteriophage  are  extremely  toxic,  possessed  of  a 
great  vitality,  inagglutinable  by  a  specific  serum,  and  actively 
ferment  maltose.4 

B.  dysenteriae  Hiss 

An  anti-Hiss  bacteriophage  is  frequently  found  in  the  nor- 
mal intestine.  A  bacteriophage  showing  an  activity  for  any 
member  of  the  colon-typhoid-dysentery  group  frequently  shows 
a  virulence,  more  or  less  pronounced,  for  the  Hiss  bacillus. 

B.  dysenteriae  Hiss  represents  a  homogeneous  species  from  the 
point  of  view  of  bacteriophagous  activity. 

Secondary  cultures  reinoculated  into  litmus  sugar  media  do 
not  ferment  the  sugars  in  the  same  way  as  do  normal  bacilli. 
Media  containing  glucose,  maltose,  and  mannite  become  acid 
after  ten  days;  those  containing  lactose,  levulose,  saccharose, 
and  also  glycerine  remain  alkaline.  After  a  month  the  lactose, 
saccharose  and  levulose  media  remain  alkaline.  Secondary  cul- 
tures, and  also  mixed  cultures,  give  the  indol  reaction  but  do  not 
react  on  either  neutral  red  or  lead  acetate.  The  resistant  bacilli 
are  inagglutinable,  have  a  high  viability,  and  are  more  virulent 
for  man.  In  Part  II  of  this  monograph  we  will  consider  a  case 
of  B.  dysenteriae  Hiss  septicemia  in  which  the  bacillus  was  re- 
sistant to  the  action  of  the  bacteriophage. 

B.  dysenteriae  Flexner 

The  anti-Flexner  bacteriophage  is  found  in  the  normal  intestine 
of  vertebrates  as  frequently  as  are  the  other  strains  of  bacterio- 
phage.5 

4  Pottevin  has  shown  that  the  normal  Shiga  bacillus  definitely,  although 
somewhat  weakly,  ferments  maltose.  Resistant  bacilli  ferment  the  sugar 
much  more  energetically. 

6  The  presence  of  an  active  bacteriophage  in  pathologic  conditions  is 
not  considered  here.  This  phase  will  be  discussed  in  Part  II  of  this  text. 


106  THE  BACTERIOPHAGE 

All  strains  isolated,  active  for  the  Flexner  bacillus,  have  like- 
wise been  active  for  B.  coli,  although  with  some,  activity  for 
other  varieties  of  dysentery  bacilli  was  lacking. 

With  reference  to  the  bacteriophage,  Flexner  bacilli  constitute 
a  homogeneous  species.  Resistant  bacilli  ferment  glucose,  levu- 
lose,  maltose,  and  mannite.  They  do  not  ferment  lactose,  do 
not  blacken  lead  acetate  in  an  agar  medium,  and  do  not  react  on 
neutral  red.  They  form  indol.  They  are  inagglutinable  by  a 
specific  serum  and  possess  a  high  viability. 

The  atypical  character  of  certain  strains  of  B.  dysenteriae  when 
freshly  isolated  from  the  organism  may  surely  be  ascribed  to 
their  resistance  to  the  bacteriophage.  Elsewhere  we  will  con- 
sider a  typical  case.  Furthermore,  this  observation  is  of  general 
significance,  applicable  not  to  dysentery  bacilli  alone. 

B.  dysenteriae  "X" 

During  the  course  of  these  investigations  a  very  great  number 
of  specimens  of  feces,  derived  from  patients  with  intestinal  dis- 
turbances, have  been  examined.  And  in  many  cases  of  gastro- 
enteritis in  adults  as  well  as  in  infants  a  bacillus  having  the  fol- 
lowing characteristics  has  been  isolated: 

When  inoculated  on  litmus  sugar  agar  media  it  fails  to  ferment 
any  of  the  sugars  tested  (lactose,  glucose,  levulose,  saccharose, 
maltose,  mannite,  galactose).  It  causes  no  change  in  lactose 
and  maltose  Barsiekow  medium,  but  this  medium  containing 
glucose  and  mannite  is  turned  red.  It  is  agglutinated  by  con- 
valescent serum  in  titres  of  1:100  to  1:500,  is  not  agglutinated 
by  anti-Flexner  or  anti-Shiga  sera.  With  a  serum  which  agglu- 
tinates the  Hiss  strain  to  1:2500  the  "X"  strain  is  agglutinated 
in  dilutions  of  1:200.  It  is  non-motile,  is  morphologically  like 
the  other  dysentery  organisms,  is  Gram-negative,  and  is  toxic 
for  rabbits. 

Several  strains  of  bacteriophage  active  for  this  bacillus  have 
been  isolated.  This  bacteriophage  is  constantly  present  in  the 
intestine  in  convalescents  who  have  shown  B.  dysenteriae  "X" 
in  their  stools  during  the  infection.  Strains  have  also  been 
recovered  from  the  intestinal  tracts  of  healthy  animals,  both 
man  and  other  animals.  The  "X"  bacillus  constitutes  a  homo- 
geneous species  as  regards  the  bacteriophage. 


VIEULENCE   OF  THE   BACTERIOPHAGE  107 

Certain  strains  of  bacteriophage  active  for  B.  dysenteriae  "X" 
were  likewise  active  for  other  species  of  dysentery  bacilli,  others 
were  virulent  for  only  one  or  two  among  them.  When  maintained 
for  several  generations  at  the  expense  of  B.  dysenteriae  "X" 
they  almost  completely  lose  their  activity  for  other  dysentery 
organisms. 

B.  coli 

An  anti-coli  bacteriophage  is  extremely  frequent  in  the  feces 
of  normal  vertebrates  and  invertebrates,  but  only  exceptionally 
is  it  found  possessed  of  any  considerable  virulence.  On  the  other 
hand,  in  recovery  from  the  most  varied  pathologic  conditions 
very  active  strains  can  be  isolated.  B.  coli,  particularly  when 
recently  isolated,  constitutes  a  heterogeneous  species  as  regards 
the  bacteriophage.  In  the  presence  of  a  bacteriophage  possess- 
ing a  very  high  virulence  for  certain  coli  strains,  other  races  are 
hardly  touched,  some  are  even  absolutely  refractory.  When 
taken  from  the  body,  B.  coli  always  shows  a  degree  of  resistance. 
In  the  intestine  it  forms  with  the  bacteriophage  a  mixed  culture. 
On  artificial  culture  media  the  resistance  decreases  very  slowly 
with  successive  transfers. 

With  a  colon  organism  of  maximum  resistance,  that  is,  one 
which  is  completely  refractory,  the  colonies  on  agar  are  large, 
white,  fluent,  exactly  like  those  of  the  bacillus  of  Friedlander. 

B.  typhosus 

Quite  frequently  a  strain  of  bacteriophage  showing  a  slight 
activity  for  B.  typhosus  can  be  isolated  from  the  normal  intestine. 
The  isolation  of  a  very  active  strain  is  exceptional  since  such  are 
found  only  in  convalescents.  A  single  strain  of  bacteriophage 
may  show  a  very  great  variation  in  virulence  for  different  races 
of  B.  typhosus,  certain  races  being  entirely  resistant  to  a  given 
bacteriophage  although  they  may  be  very  susceptible  to  other 
strains  of  the  bacteriophage.  In  such  a  case  there  is  a  natural 
resistance,  a  true  natural  immunity,  a  condition  which  can  be 
demonstrated  not  only  for  the  typhoid  bacillus  but  also  for  B. 
coli,  the  paratyphoid  bacilli,  B.  proteus,  etc.  It  is  because  of  such 
reactions  that  these  organisms  are  spoken  of  as  belonging  to 


108  THE   BACTERIOPHAGE 

species  which  are  not  homogeneous  as  regards  the  bacteriophage. 
Typhoid  bacilli  may  acquire,  in  vivo  or  in  vitro,  a  resistance  to 
the  action  of  the  bacteriophage,  that  is,  they  may  possess  an 
acquired  immunity  (this  must  not  be  confused  with  natural  im- 
munity) and  they  become  inagglutinable  as  well  as  possessed  of 
an  enhanced  virulence  for  experimental  animals.  As  has  been 
shown  for  B.  dysenteriae,  repeated  culturing  on  agar  progressively 
lowers  the  resistance  to  the  bacteriophage,  and  coincidently 
restores  agglutinability. 

B.  paratyphosus  A 

A  bacteriophage  showing  virulence  for  this  bacillus  is  relatively 
frequent  in  the  normal  intestine.  As  regards  the  action  of  the 
bacteriophage,  B.  paratyphosus  A  strains  form  a  more  homogeneous 
species  than  do  the  typhoid  bacilli.  Just  as  with  B.  typhosus, 
the  paratyphoid  A  organisms  may  acquire  a  resistance  to  the 
bacteriophage,  may  become  inagglutinable,  and  may  show  an 
increased  virulence. 

B.   paratyphosus  B 

A  bacteriophage  for  this  organism  is  very  frequent  in  normal 
stools.  The  resistance  of  B.  paratyphosus  B  places  this  bacillus 
intermediary  between  B.  typhosus  and  B.  paratyphosus  A.  Re- 
sistant bacteria  are  inagglutinable  and  are  of  high  virulence. 
Mixed  colonies  on  agar,  in  which  the  bacterium  has  acquired  a 
high  resistance  for  the  bacteriophage,  present  a  viscid  appearance 
resembling  B.  Friedldnder. 

Salmonella   (hog  cholera) 

One  strain  of  bacteriophage  active  for  this  organism  has  been 
isolated  from  a  normal  man.  When  derived  from  the  body  it 
possessed  an  average  virulence. 

B.  typhi  murium 

The  anti-paratyphoid  B  strains  of  the  bacteriophage  are  some- 
times endowed  with  virulence  for  B.  typhi  murium.  Very  active 
strains  have  been  isolated  from  the  intestinal  tracts  of  white  and 


VIRULENCE   OF  THE   BACTERIOPHAGE  109 

gray  rats  which  were  rendered  experimentally  resistant  to  the 
disease  caused  by  the  ingestion  of  cultures  of  the  bacillus.  The 
resistant  bacilli  are  very  virulent  and  can  be  used  for  the  destruc- 
tion of  gray  rats,  a  large  proportion  of  which  resist  the  action  of 
the  ordinary  virus.  It  is  possible  that  human  infection  may  be 
feared  because  of  this  increased  virulence. 

The  transitory  appearance  of  such  a  bacteriophage  in  the 
blood  of  several  infected  white  rats  has  been  demonstrated.  Such 
rats  were  resistant  to  infection. 

B.  proteus 

Two  strains  of  bacteriophage  very  active  for  this  bacillus  have 
been  isolated  from  the  stools  of  two  infants  having  a  gastro- 
enteritis. The  virulence  of  these  strains  was  tested  against  a 
dozen  strains  of  B.  proteus  of  different  origins.  Only  three  of 
the  strains  tested  were  affected  by  these  strains  of  the  bacterio- 
phage, the  same  three  in  both  cases.  The  other  nine  proteus 
strains  were  non-susceptible.  Included  in  this  last  group  were 
two  strains  of  B.  proteus  Xi*. 

The  lysate  secured  through  the  interaction  of  a  bacteriophage 
on  a  proteus  suspension,  is,  immediately  after  the  lysis,  extremely 
toxic  for  rabbits.  Indeed,  they  are  killed  within  a  few  hours  by 
the  subcutaneous  injection  of  but  half  of  a  cubic  centimeter. 
After  ten  days  the  lysate  loses  its  toxicity. 

B.  gallinarum  (Klein);  B.  gallinarum  (Moore);  B.  paragallinarum 

Characterization  of  these  bacilli  will  be  reserved  for  the  chapter 
in  which  avian  typhosis  is  discussed.  With  the  exception  of 
pathogenicity  for  man  B.  gallinarum  presents  all  the  characteris- 
tics of  B.  typhosus,  including  agglutinability  to  the  titre  of  the 
serum  with  an  anti-typhoid  serum.  There  are,  as  we  will  see, 
at  least  three  different  species  of  paragallinarum  organisms. 

The  bacteriophage  active  for  B.  gallinarum  is  not  effective 
with  all  species  of  paragallinarum,  nor  is  the  anti-paragallinarum 
bacteriophage  active  for  the  other  races.  B.  gallinarum  is  a  very 
homogeneous  species.  The  anti-gallinarum  bacteriophage  is 
constantly  present  in  the  intestinal  tracts  of  fowls  which  resist 


110  THE   BACTEEIOPHAGE 

infection.  It  has  been  isolated  from  the  blood  of  three  fowls 
which  were  recovering  from  the  infection.  Outside  of  epizootic 
foci  it  has  been  found  in  the  intestine  of  healthy  animals. 

Bad.   diphtherias 

Two  strains  of  bacteriophage  active  for  only  atoxic  strains  of 
Bact.  diphtheriae  have  been  isolated  from  the  feces  of  two  horses 
immunized  by  the  injection  of  cultures  of  diphtheria  bacilli. 
This  observation  is  only  mentioned.  Lack  of  opportunity  has 
prevented  further  examination  of  these  strains,  a  study  which 
certainly  offers  much  of  interest. 

Staphylococcus 

A  bacteriophage  active  for  the  Staphylococcus  has  been  isolated 
under  the  following  circumstances.  A  guinea  pig  bit  my  left 
index  finger  and  on  the  next  day  an  inflammation  appeared  which 
persisted  for  three  days.  On  the  fourth  day  there  was  an  accumu- 
lation of  pus,  of  which  about  ten  drops  were  secured.  When 
planted  directly  upon  agar  there  developed  one  colony  of  Sta- 
phylococcus albus  and  six  of  Staphylococcus  aureus.  The  remainder 
of  the  pus  was  mixed  with  twenty  cubic  centimeters  of  bouillon 
and  placed  in  the  incubator  for  24  hours,  then  filtered  through 
infusorial  earth  and  a  bougie.  After  five  passages  at  the  expense 
of  Staphylococcus  albus  lysis  was  secured.  At  first  the  bacterio- 
phage isolated  did  not  show  any  activity  in  vitro  for  Staphylococ- 
cus aureus.  It  has  been  possible  to  develop  a  virulence  for  this 
organism  only  after  about  fifty  passages  in  a  mixed  Staphylococ- 
cus aureus  and  Staphylococcus  albus  suspension,  following  the 
technic  previously  indicated  for  the  enhancement  of  the  latent 
virulence  of  an  anti-staphylococcus  bacteriophage  toward  B. 
dysenteriae  Shiga. 

Bacterium  of  barbone 

About  thirty  strains  of  bacteriophage  for  this  organism  have 
been  isolated,  of  which  twelve  were  extremely  active.  Their 
activity  was  comparable  when  tested  against  different  bacterial 
strains,  some  derived  from  Italy,  others  f rom  Indo-China.  We 


VIRULENCE   OF  THE   BACTEEIOPHAGE  111 

will  return  to  a  consideration  of  this  bacteriophage  later,  in  the 
chapter  dealing  with  barbone  (hemorrhagic  septicemia  of  the 
buffalo) . 

When  isolated  from  the  body  all  the  strains  presented  an  average 
or  feeble  virulence  toward  the  different  intestinal  bacteria.  After 
about  a  dozen  passages  at  the  expense  of  the  bacterium  of  bar- 
bone  these  accessory  virulences  became  markedly  attenuated. 

B.  pestis 

Twelve  strains  of  bacteriophage  active  for  B.  pestis  have  been 
isolated.  Eleven  of  these  were  secured  from  the  excreta  of  rats 
in  the  different  villages  of  Indo-China  where  plague  was  epidemic. 
The  twelfth  was  derived  from  the  feces  of  a  patient  convalescent 
from  plague.  This  is  the  only  strain  which  has  been  maintained. 
This  strain  was  also  active  for  the  bacillus  of  pseudotuberculosis 
of  guinea  pigs. 

Bacillus  of  flacherie 

The  bacillus  in  question  was  isolated  from  the  bodies  of  silk- 
worms which  had  died  of  a  disease  presenting  the  characteristics 
of  flacherie  in  the  breeding  establishments  in  Indo-China.  The 
bacteriophage  is  frequent  in  the  intestine  of  the  healthy  worms 
among  a  contaminated  stock.  The  activity  of  this  bacteriophage 
was  the  same  for  each  of  the  three  strains  of  the  bacillus  tested, 
isolated  from  three  different  breeding  places. 

B.  subtilis 

One  strain  of  bacteriophage  active  for  this  bacillus  was  secured 
in  the  stools  of  a  patient  with  dysentery.  Having  but  rarely 
tested  the  virulence  of  the  bacteriophage  toward  B.  subtilis  it  is 
impossible  to  say  if  this  virulence  is  frequent  or  exceptional. 

Vibrio  cholerae 

Among  about  one  hundred  cases  of  cholera  studied  in  Indo- 
China  it  was  possible  to  observe  but  one  following  recovery.  In 
this  last,  in  spite  of  daily  examination  of  stools,  in  but  a  single 
specimen  taken  at  the  beginning  of  convalescence  has  a  bacterio- 


112  THE   BACTERIOPHAGE 

phage  active  for  the  vibrio  been  found.  This  gave  about  fifty 
plaques  when  planted  on  agar.  In  spite  of  many  attempts  it 
has  been  impossible  to  cultivate  it  by  serial  transfers.  None 
of  the  fatal  cases  yielded  a  bacteriophage. 

The  diversity  of  the  bacterial  types  against  which,  up  to  the 
present  time,  virulent  strains  of  bacteriophage  have  been  iso- 
lated, suggests  the  idea  that  the  activity  of  the  bacteriophage 
may  be  manifested  toward  any  bacterial  species  whatsoever. 


CHAPTER  IV 

THE  BACTERIOPHAGOTJS  ULTRAMICROBE 

Morphology.  Viability.  Susceptibility  to  Different  Substances.  Unicity 
of  the  Bacteriophage.  Lysins  of  the  Bacteriophage.  Opsonic  Power 
of  the  Lysins. 

MORPHOLOGY 

The  bacteriophagous  ultramicrobe  is  of  extreme  tenuity.  In 
a  medium  containing  the  bacteriophage  the  ultramicroscope 
reveals  only  some  very  minute  brilliant  points.  Probably  each 
of  these  points  represents  an  ultramicrobe,  particularly  since 
their  abundance,  in  greater  or  lesser  numbers,  corresponds  some- 
what with  the  counts  made  upon  agar.  Its  tenuity  is  such  that 
a  medium  containing  several  thousand  million  ultramicrobes 
per  cubic  centimeter  appears  perfectly  limpid.  The  ultramicrobe 
is,  however,  resident  in  a  definite  mass,  since  each  element  is 
deposited  on  agar  in  distinct  points  and  this  mass  must  be  appre- 
ciable since  the  ultramicrobes  spontaneously  sediment  in  the 
course  of  time. 

Experiment  XXX.  A  culture  of  an  anti-dysentery  bacteriophage  is 
filtered  through  a  bougie  and  allowed  to  stand  without  moving  in  a  cup- 
board for  eleven  months.  At  the  end  of  this  time,  specimens  of  the  cul- 
ture from  the  surface  and  from  the  bottom  of  the  tube  are  taken  with 
capillary  pipettes. 

The  count  of  the  superficial  layers  showed  280,000,000  per  cubic  centi- 
meter. 

The  count  of  the  deeper  layers  showed  2,900,000,000  per  cubic  centi- 
meter. 

The  ultramicrobe  can  be  sedimented,  although  incompletely, 
by  centrifugation  at  very  high  speed. 

Experiment  XXXI.  Twenty-five  cc.  of  the  bacteriophage  (antidysen- 
tery)  are  filtered  through  a  bougie  and  are  centrifuged  in  a  Jouan  appara- 
tus for  30  minutes  at  12,000  revolutions  per  minute.  Counts  show  the 
following: 

113 


114  THE   BACTERIOPHAGE 

per  cubic  centimeter 

Before  centrifugation 1,750,000,000 

Surface 50,000,000 


)n\Bottom 3,700,000,000 

Dialysis  through  collodion  membranes  of  various  permeabilities 
gives  a  rough  approximation  of  the  size  of  the  bacteriophagous 
ultramicrobe.  As  a  test,  one  part  of  horse  serum  was  mixed  with 
three  parts  of  a  culture  of  the  bacteriophage,  and  the  mixture  was 
subjected  to  dialysis.  Whenever  the  albumin  passed  through  the 
filter  the  bacteriophage  passed  also,  and  when  the  permeability 
was  such  that  the  albumin  was  held  back,  so  also  was  the  bac- 
teriophage. 

The  bacteriophage  then,  passes  through  when  the  molecule  of 
serum  albumin  passes  and  is  retained  when  the  latter  is  held  back. 
It  remains  for  physicists  to  more  exactly  determine  its  true  size, 
and  this  determination  will  be  of  more  interest  since  the  bac- 
teriophage is  the  only  ultramicrobe  with  which  such  measurement 
is  actually  possible,  since  it  is  the  only  one  where  the  elements 
can  be  counted.  Thus,  it  may  serve  to  clear  up  an  important 
point  touching  the  constitution  of  organized  matter.  If  one 
calculates  the  ultramicrobe  as  being  one  one-hundredth  of  a 
micron  in  diameter,  it  ought  to  contain  about  twenty  molecules 
of  albumin  and  five  or  six  atoms  of  sulfur.  Physicists  have  de- 
termined the  size  of  the  pores  in  the  most  dense  collodion  mem- 
branes as  being  not  greater  than  two  millionths  of  a  micron.  But 
the  ultramicrobe  of  avian  plague  penetrates  such  a  membrane. 
Each  element  can  not  be  greater  than  one  five-thousandth  of  a 
micron  in  diameter,  hence  it  would  be  composed  of  one-tenth  of 
a  molecule  of  albumin.  On  the  other  hand,  an  ultramicrobe  is 
indeed  a  markedly  complex  organism,  capable  of  adaptation, 
possessing  the  faculty  of  secreting  toxins, — the  diastases, — having 
in  a  word,  the  characteristics  of  living  matter.  This  in  itself 
implies  a  relatively  complex  organism.  We  find  ourselves,  then, 
cornered  by  an  absurdity,  for  it  is  impossible  to  conceive  of  a 
complex  organism  formed  of  a  single  molecule,  much  less  of  the 
tenth  part  of  one.  It  will  be  much  more  simple  to  admit  that  it 
is  impossible  to  understand  under  what  aspect  life  is  present  in 
the  ultramicrobe  and  under  what  form  the  matter  composing  it 
exists. 


BACTERIOPHAGOTJS   ULTRAMICROBE  115 

It  is  only  since  the  discovery  of  the  bacteriophage  that  it  has 
been  possible  to  affirm  that  each  ultramicrobe  is  a  material  mass 
capable  of  multiplication  in  the  form  of  like  masses.  Thanks  to 
it,  our  ideas  regarding  viruses  have  acquired  some  degree  of  pre- 
cision. The  study  of  the  bacteriophage  by  physicists  would 
offer  findings  of  extreme  interest,  for  it  is  the  only  virus  demon- 
strated by  experiment  to  exist  in  particulate  form,  and  with  this 
alone  is  it  actually  possible  to  fix  dimensions,  thanks  to  the  possi- 
bility of  recognizing  the  number  of  elements  present  in  a  liquid. 

VITALITY 

The  bacteriophagous  ultramicrobe  is  extremely  resistant  toward 
the  majority  of  destructive  agents,  a  property  which  it  shares, 
moreover,  with  other  ultramicrobes. 

The  vitality  is  very  great.  Filtrates  or  cultures  containing 
the  bacteriophage  are  still  active  after  six  years  when  preserved 
in  a  sealed  tube.  However,  not  all  of  the  germs  present  in  a 
culture  show  the  same  degree  of  resistance.  After  preservation 
for  four  years  a  culture  which  originally  contained  two  thousand 
million  ultramicrobes  per  cubic  centimeter  contains  only  about 
one  hundred  millions  of  living  organisms.  Such  vitality  is  not 
exceptional,  for  certain  bacteria,  not  spore-forming,  show  a  re- 
sistance of  the  same  order.  For  example,  cultures  of  B.  coli  are 
still  cultivable  after  ten  years  or  so,  and  here  also  the  different 
bacilli  of  a  culture  do  not  offer  the  same  resistance,  for  the  num- 
ber of  those  which  survive  becomes  smaller  and  smaller  with 
time. 

If  a  culture  of  the  bacteriophage  is  allowed  to  evaporate  slowly 
at  room  temperature  it  is  found  that  living  germs  may  be  found 
in  the  few  drops  of  syrupy  fluid  remaining  in  the  bottom  of  the 
tube.  Indeed,  certain  bacteria  act  in  the  same  way.  On  the 
contrary,  living  organisms  are  no  longer  to  be  found  after  twelve 
months  in  glucose  bouillon  cultures,  although  they  may  still  be 
alive  in  lactose  bouillon. 

In  fecal  material  preserved  at  room  temperature  in  sealed 
tubes  for  thirty-four  months  (September,  1915,  to  July,  1918) 
one  may  recover  the  living  bacteriophage,  as  active  as  at  the 
beginning.  This  experiment  has  been  performed  successfully 
with  four  specimens  of  feces  from  convalescent  cases  of  dysentery. 


116  THE  BACTEBIOPHAGE 

In  a  dry  state  the  bacteriophage  is  resistant  for  a  long  time. 
A  fragment  of  sterile  filter  paper  is  saturated  with  a  drop  of  a 
bacteriophage  culture  (anti-dysentery),  dried  in  the  air,  and  pre- 
served in  a  sealed  tube  for  six  months  at  room  temperature.  After 
this  time  the  piece  of  paper  is  introduced  into  a  suspension  of 
B.  dysenteriae  and  normal,  although  delayed,  lysis  is  obtained. 
The  ultramicrobes  have,  therefore,  survived.  Another  ultrami- 
crobe,  that  of  the  tobacco  mosaic,  has  the  same  property.  It 
remains  alive  for  two  years  in  the  dried  leaves.  It  is  indeed, 
unnecessary  to  search  for  examples  of  bacteria  as  resistant  as 
the  ultramicrobes.  The  cocco-bacillus  of  locusts  is  a  non-sporu- 
lating  bacillus,  but  in  the  cadavers  of  locusts  dead  of  the  disease 
which  it  incites  in  these  insects  (cadavers  dried  over  sulfuric 
acid,  pulverized,  and  preserved  in  sealed  tubes  for  three  years). 
I  have  shown  that  the  cocco-bacillus  remains  alive  and  virulent, 
for  this  powder,  seeded  into  bouillon,  gives  normal  cultures  viru- 
lent for  the  locust. 

SUSCEPTIBILITY   TO   DIFFEKENT   AGENTS1 

Physical  agents:  Effect  of  temperature 

At  the  beginning  of  my  experiments  I  stated  that  the  tempera- 
ture of  destruction  of  the  bacteriophage  is  about  65°C.  Shortly 
after  this  Kabeshima  in  an  early  report  cited  70  to  75°C.,  and  in 
a  later  note  70°C.  Very  recently  Gratia  and  Jaumain  have  noted 
that  the  lethal  temperature  showed  considerable  variability; 
61  or  62°C.  for  the  lytic  principle  acting  on  the  staphylococcus 
and  65°C.  for  that  acting  on  the  colon  bacillus. 

For  testing  this  point  I  had  taken  as  a  criterion  the  ability  or 
inability  of  material  subjected  to  different  temperatures  to  pro- 
duce lysis  of  a  bacterial  suspension.  Moreover,  this  has  been 
the  method  adopted  by  the  other  investigators  who  have  consid- 
ered this  question.  In  view  of  these  contradictory  results,  the 
effect  of  temperature  has  been  reconsidered,  taking  as  a  criterion, 
not  lysis  of  a  suspension  in  a  fluid  medium,  but  the  action  of  a 
culture  on  solid  media,  a  procedure  much  more  delicate. 

1  The  experiments  dealing  with  the  effects  of  temperature  have  been 
made  in  collaboration  with  E.  Pozerski. 


BACTERIOPHAGOUS  ULTRAMICROBE  117 

Experiment  XXXII.  In  the  following  experiments  the  culture  of  bac- 
teriophage  under  test,  previously  filtered  through  a  bougie,  is  taken  up  in 
capillary  pipettes,  sealed  at  both  ends,  and  completely  submerged  in  a 
water-bath  maintained  at  the  temperatures  indicated  in  each  experiment. 
In  each  series  of  experiments  8  tubes  with  culture  are  maintained  for 
thirty  minutes  at  temperatures  of  60,  62,  64,  66,  68,  70,  72,  and  75 °C. 

Anti-Shiga  bacteriophage 

Two  drops  of  the  culture  from  tubes  maintained  at  60,  62,  64,  and  66°C., 
when  introduced  into  suspensions  of  Shiga  bacilli,  cause  complete  lysis 
in  less  than  fourteen  hours.  The  tests  repeated  with  a  second  strain  of 
Shiga  bacilli  give  identical  results.  The  bacteriophage  heated  to  68  and 
70°C.  causes  lysis  with  one  strain  of  Shiga  bacilli  but  not  with  the  other. 
When  heated  to  72  and  75°C.  the  bacteriophage  fails  to  cause  lysis. 

One  drop  of  each  of  these  suspensions,  which  had  received  the  bacterio- 
phage cultures  previously  maintained  at  68,  70,  72  and  75 °C.,  and  which 
had  not  been  submitted  to  lysis,  are  planted  on  slant  agar.  After  incuba- 
tion, all  of  the  cultures,  except  the  last,  which  is  normal,  show  plaques 
characteristic  of  the  presence  of  the  bacteriophage. 

Serial  passages  may  be  effected,  thus  permitting  the  enhancement  in 
virulence  of  the  bacteriophage  attenuated  by  the  action  of  temperature. 
After  two  such  passages,  with  the  ultramicrobe  heated  to  68  and  70°C., 
and  after  three  passages  with  that  which  was  heated  to  72°C.,  lysis  in 
liquid  media  is  obtained. 

Comparable  experiments  have  demonstrated  that  the  bacteriophagous 
ultramicrobes  active  for  B.  dysenteriae  Flexner,  B.  dysenteriae  Hiss,  B.  coli, 
and  B.  paratyphosus  B,  act  in  a  quite  similar  manner.  With  the  bacterio- 
phage active  for  B.  paratyphosus  A  attenuation  begins  at  about  64°C.  (at 
least  with  the  strain  tested).  With  that  virulent  for  B.  typhosus  attenua- 
tion is  already  apparent  at  about  62°C.  In  all  cases,  when  heated  to  75°C. 
the  bacteriophage  is  completely  inactive,  either  actually  destroyed  or 
attenuated  to  such  an  extent  that  its  presence  can  no  longer  be  detected. 
In  all  these  instances  the  bacteriophage  shows  a  recuperative  power,  the 
virulence  being  restored  when  the  temperature  to  which  the  virus  has  been 
subjected  is  not  higher  than  72°C. 

Anti-staphylococcus  bacteriophage 

Attenuation  of  this  bacteriophage  is  already  manifest  after  heating  to 
60°C.  Subcultures  of  suspensions  which  have  not  been  lysed  show  that  it 
is  a  simple  attenuation,  for,  even  with  suspensions  inoculated  with  a  bac- 
teriophage previously  held  at  72°C.  for  thirty  minutes,  plaques  are  obtained 
characteristic  of  the  presence  of  an  active  bacteriophage.  Moreover,  two 
passages  suffice  to  restore  the  original  virulence  to  cultures  heated  to  62, 
64,  66,  and  68°C.  After  heating  at  70  and  72°C.  the  attenuation  of  virulence 
does  not  disappear  until  after  six  passages.  When  heated  to  75°C.  the 
bacteriophage  is  deprived  of  all  activity. 


118  THE   BACTEKIOPHAGE 

It  may  be  concluded  from  these  experiments  that  all  strains 
of  the  bacteriophage  react  to  temperature  in  the  same  manner. 
When  heated  above  60°C.  they  are  attenuated  more  or  less  rapidly 
according  to  the  bacterial  species  upon  which  they  operate.  All 
are  completely  killed,  or  at  least  paralyzed,  at  about  75°C. 

Chemical  agents 

The  bacteriophagous  ultramicrobe  will  attack  bacteria  either 
in  the  presence  or  absence  of  oxygen,  or  indeed  in  an  atmosphere 
of  either  nitrogen  or  hydrogen. 

Antiseptics 

It  is  interesting  to  study  the  action  of  antiseptics  for  these 
substances  do  not  act  in  the  same  manner  on  certain  ultramicrobes 
as  on  ordinary  bacteria.  While  very  sensitive  to  the  action  of 
certain  antiseptics,  they  are  very  resistant  to  others. 

A  culture  of  anti-dysentery  bacteriophage  in  physiological 
saline2  is  distributed  into  four  tubes.  One  serves  as  a  control. 
The  second  receives  mercuric  chlorid  to  a  concentration  of  1:200, 
the  third  receives  copper  sulfate  to  a  concentration  of  1:100, 
and  to  the  fourth  phenol  is  added  in  a  concentration  of  1 : 100. 
After  contact  with  these  substances  for  three  days  the  bacterio- 
phage is  living  in  all  the  tubes.  After  four  days  it  is  killed  in  the 
tubes  containing  the  mercuric  chlorid  and  the  copper  sulfate. 
After  seven  days  it  is  killed  by  the  carbolic  acid.  It  remains 
alive  in  the  control  tube. 

It  is  not  killed  after  contact  for  a  week  in  a  fluid  saturated 
with  essence  of  thyme  or  of  cloves,  but  its  lytic  action  is  not  mani- 
fested there.  The  same  results  are  secured  in  media  containing 
chloroform  or  sodium  fluoride  (Bablet). 

Eliava  and  Pozerski  have  determined  with  precision  the  lethal 
limits,  in  24  hours,  of  concentrations  of  free  H  and  OH  ions.  The 
zone  compatible  with  life  lies  between  pH  2.5  and  8.54,  corre- 
sponding approximately  to  an  acid  1/160  N,  and  a  base  1/260 
N,  whatever  may  be  the  acid  or  alkali  tested. 

2  Bouillon  is  not  suitable  for  this  work  because  of  the  precipitates  which 
form  upon  the  addition  of  certain  antiseptics.  These  give  rise  to  entirely 
false  results. 


BACTERIOPHAGOUS   ULTRAMICROBE  119 

It  is  difficult  to  make  a  direct  comparison  with  the  limits  of 
resistance  of  other  micro-organisms,  the  bacteriophagous  ultra- 
microbe  is  at  present  the  only  one  for  which  such  determinations 
have  been  made.3 

The  action  of  glycerine  is  very  interesting.  The  bacteriophage 
remains  alive  for  at  least  two  years  in  a  fluid  composed  of  equal 
parts  of  glycerine  and  bouillon  or  physiological  saline.  A  sus- 
pension of  bacteria  in  such  a  medium,  a  medium  in  which  the 
bacteria  are  unable  to  reproduce,  is  lysed  as  perfectly  as  in  or- 
dinary bouillon,  yet  in  a  higher  concentration  of  glycerine  the 
bacteriophage  is  destroyed.  Bablet  has  in  fact  shown  that 
when  0.5  cc.  of  bacteriophage  is  added  to  9.5  cc.  of  glycerine  the 
ultramicrobe  is  killed  in  six  days.  It  may  be  well  to  recall  that 
glycerine  constitutes  the  best  medium  for  the  conservation  of  the 
toxins  and  diastases.  The  other  known  ultramicrobes  resist, 
in  general,  the  action  of  glycerine. 

We  have  seen  that  a  suspension  in  a  glycerine  medium  may  be 
lysed  by  the  bacteriophage,  and,  as  always,  the  lysed  culture 
becomes  a  culture  of  the  bacteriophage.  If  we  allow  such  a  cul- 
ture to  evaporate  slowly  at  room  temperature  we  will  finally  have 
a  residue  composed  of  glycerine,  all  the  water  being  evaporated. 
Under  such  conditions,  the  bacteriophage  becomes  adapted  to 
its  environment  and  remains  alive  in  the  glycerine  residue,  al- 
though it  is  killed  if  transferred  directly  from  a  bouillon  culture 
to  concentrated  glycerine.  Many  instances  are  known  of  the 
adaptation  of  bacteria  to  antiseptics;  and  adaptation  is  a  func- 
tion of  living  matter. 

8 1  may  cite,  for  example,  the  following  findings  which  have  been 
reported,  not  on  the  zone  of  life,  but  on  the  zone  of  growth. 

G.  Dernby  (Ann.  de  1'Inst.  Pasteur,  1921,  35,  277)  gives  the  following 
figures: 

Staphylococcus pH  4.8  to  8.1 

B.  subtilis pH  4.5  to  8. 5 

B.  proteus pH  4.4  to  8.4 

B.  coli pH  4. 4  to  7.8 

The  bacteriophage  is,  therefore,  extremely  sensitive  to  the  action  of 
bases  and  acids,  since  its  fatal  limit  of  alkalinity  is  the  same  as  the  limit 
for  growth  of  ordinary  bacteria ;  its  fatal  acid  limit  is  not  far  distant.  It  is, 
therefore,  more  sensitive  than  bacteria  to  concentrations  of  free  H  and  OH 
ions.  This  is  further  evidence  of  its  living  nature. 


120  THE  BACTERIOPHAGE 

Eliava  and  Pozerski  have  shown  that  the  neutral  salts  of  qui- 
nine exert  an  antiseptic  action  on  the  bacteriophage,  in  three  per 
cent  solution  killing  it  in  thirty  minutes,  in  one  per  cent,  in  a  few 
hours.  Emetine  hydrochlorate  and  saponine  in  the  same  con- 
centrations are  without  action.  This  susceptibility  to  the  anti- 
septic action  of  quinine  is  singular  in  a  germ  which  is  otherwise 
relatively  resistant  to  antiseptic  activities.  It  is  hardly  possible 
to  deduce  that  the  bacteriophage  is  protozoan  in  nature,  for 
quinine  exerts  antiseptic  properties  toward  many  bacterial  species, 
although  on  the  contrary,  it  is  without  action  on  the  diastases 
and  toxins. 

When  a  culture  is  treated  with  acetone  the  albuminoid  materials 
of  the  bouillon  are  thrown  out  of  solution,  and  this  precipitate 
encloses  the  bacteriophagous  virus.  The  greater  portion  of  the 
virus  is,  however,  destroyed.  The  bacteriophage  reacts  like  the 
spore-forming  bacteria,  which  are  found,  living,  in  the  precipi- 
tate. In  this  connection  a  curious  thing  has  been  noted. 
Ordinarily  acetone  is  considered  a  sterile  fluid,  but  it  is  not 
necessarily  so,  since  it  has  been  found  that  several  containers  of 
acetone  have  been  contaminated  by  B.  subtilis. 

Alcohol  gives  the  same  precipitate,  but  the  bacteriophage,  con- 
trary to  that  which  happens  with  the  virus  of  the  tobacco  mosaic, 
is  killed  in  less  than  forty-eight  hours  in  90  per  cent  alcohol. 
The  precipitate,  as  we  will  see,  contains  the  secretory  products 
of  the  bacteriophage. 

UNICITY  OF  THE  BACTERIOPHAGE 

In  the  preceding  chapters  detailed  experiments  have  been 
given  which  show  that  whatever  the  bacteria  attacked,  the  ul- 
tramicrobes  which  attack  them  belong  always  to  the  same  species. 
We  will  return  to  other  proofs  shortly.  A  single  statement, 
grouping  these  experiments  will  be  given  here. 

First.  Usually  a  single  strain  of  bacteriophage  will  attack 
several  species  of  bacteria  at  the  same  time. 

Second.  A  strain  of  the  bacteriophage,  continued  through 
more  than  a  thousand  passages  in  vitro,  always  in  conjunction 
with  the  same  bacterial  strain,  namely,  B.  dysenteriae  Shiga, 
attacks  B.  typhosus  and  B.  coli. 


BACTBRIOPHAGOUS  ULTRAMICROBE  121 

It  has  likewise  been  shown  that  a  bacteriophage  active  for  the 
staphylococcus  and  maintained  through  more  than  one  hundred 
transfers  with  this  staphylococcus  still  possessed  virulence  for 
the  dysentery  bacillus.  And  it  is  also  possible  to  effect,  in  vitro, 
the  adaptation  for  this  Shiga  bacillus  of  a  strain  of  bacteriophage 
active  only  for  Staphylococcus  aureus.  The  staphylococcus  and 
B.  dysenteriae  are  bacterial  species  but  remotely  related  and  the 
crossed  reaction  constitutes  an  irrefutable  argument  in  favor  of 
the  unicity  of  the  bacteriophage. 

Third.  An  antibacteriophagous  serum,  the  properties  of  which 
will  shortly  be  considered,  contains  an  amboceptor  specific  for 
the  bacteriophage,  as  is  demonstrated  in  the  complement  fixa- 
tion reaction  of  Bordet-Gengou,  and  this  amboceptor  is  the 
same  for  all  species  of  bacteriophage — the  anti-dysentery  bac- 
teriophage, the  anti-plague  bacteriophage  from  man,  the  anti- 
plague  bacteriophage  from  the  rat,  and  the  anti-barbone  bac- 
teriophage from  the  buffalo,  all  fix  complement  in  the  presence 
of  serum  from  a  rabbit  treated  by  repeated  injections  of  cultures 
of  the  anti-dysentery  bacteriophage.4  For  this  particular  ex- 
periment strains  of  bacteriophage  were  selected  which  failed  to 
show  a  crossed  reaction  in  vitro  with  regard  to  the  different  bac- 
teria attacked.  The  complement  fixation  reaction  is  specific 
with  respect  to  species  differentiation. 

The  proofs  of  the  unicity  of  the  bacteriophage  are  therefore 
multiple.  There  is  but  a  single  bacteriophage,  common  to  both 
man  and  animals,  capable  by  adaptation  of  acquiring  a  virulence 
toward  all  bacterial  species. 

As  we  have  seen  in  the  earlier  chapters  the  bacteriophagous 
ultramicrobe  can  not  be  cultivated  in  any  artificial  medium.  It 
is  an  obligatory  parasite,  capable  of  reproduction  only  within 
living  cells.  Moreover,  this  is  the  case  with  all  known  ultrami- 
crobes.  The  single  one  making  the  exception  to  this  rule,  the 
Asterococcus  of  pleuropneumonia  is  hardly  longer  to  be  consid- 
ered as  an  ultramicrobe,  since  it  has  been  shown  to  be  perfectly 

4  Since  the  publication  of  the  French  edition  of  this  text  Bruynoghe 
and  Maisin  have  confirmed  this  fact.  They  have  also  shown  that  fixation 
of  complement  is  also  to  be  obtained  with  an  anti-staphylococcus  bacterio- 
phage under  the  conditions  already  mentioned. 


122  THE   BACTERIOPHAGE 

visible  in  stained  preparations,  as  a  bacterium  of  minute  size. 
All  the  parasitic  ultramicrobes  are  intracellular  parasites,  for 
the  lesions  which  they  produce  are,  in  all  cases,  characterized 
by  protoplasmic  inclusions  or  alterations  in  the  nuclei  of  the 
cells.  The  bacteriophagous  ultramicrobe  differs  from  the  other 
known  ultramicrobes  only  in  its  elective  action  for  unicellular 
organisms.  The  others  act  in  multicellular  organisms. 

It  would  be  indeed  strange  that  of  all  living  organisms  the  bac- 
teria alone  should  enjoy  the  privilege  of  absolute  immunity.  Such 
an  immunity  must  have  seemed  remarkable  even  before  the  dis- 
covery of  the  bacteriophage,  before  ultramicrobes  sufficiently 
small  to  parasitize  them  had  been  recognized.  The  ultramicrobe 
is  in  diameter  certainly  2000  times  smaller  than  a  bacterium  of 
average  size,  in  volume  nearly  2000  million  times  less.  In  size, 
one  of  these  ultramicrobes  is,  to  a  bacterium,  as  the  bacterium 
is  to  a  large  fly. 

It  should  be  remembered,  however,  that  although  up  to  the 
present  time  parasitism  of  bacteria  has  not  been  recognized  we 
have  for  a  long  time  observed  and  studied  many  parasites  which 
incite  infectious  disease  among  the  protozoa.  Several  examples 
will  be  found  cited  among  the  works  of  Metchnikoff.5 

It  may  be  well  to  mention  a  study  of  Dangeard6  entitled  "Sur 
les  parasites  du  noyau  et  du  protoplasma,"  for  the  facts  disclosed 
by  this  investigator  offer  certain  analogies  to  those  presented  in 
the  preceding  chapters.  But  there  are  these  differences,  namely, 
the  parasite  of  Dangeard  attacks  a  protozoan,  and  its  dimensions 
are  such  that  it  can  be  readily  observed  microscopically  and 
therefore  classified. 

The  observations  of  Dangeard  deal  with  an  Oomycete,  Nucleo- 
phaga  amoebae  Dangeard,  which  parasitizes  the  nucleus  of  Amoeba 
verrucosa  Ehr.  The  Amoeba  verrucosa  has  a  large,  doubly-con- 
toured, spherical  nucleus,  and  also  a  nucleolus,  likewise  spherical, 
whose  diameter  is  about  two-thirds  that  of  the  nucleus.  The 
substance  of  the  nucleolus  is  very  dense  and  stains  with  great 

s  Legons  sur  la  pathologic  comparee  de  V inflammation,  Paris,  1892,  Masson 
&  Cie.  L'immunite  dans  les  maladies  infectieuses ,  Paris,  1901,  Masson  &  Cie. 

6  Sur  les  parasites  du  noyau  et  du  protoplasma,  Le  Botaniste,  1894/95, 
4, 199-248. 


BACTEEIOPHAGOUS  ULTRAMICBOBE  123 

intensity  with  various  nuclear  staining  reagents.  Between  the 
nucleolus  and  the  nuclear  membrane  is  a  space  filled  with  the 
nuclear  fluid. 

The  zoospore  of  Nuckophaga  amoeba  first  penetrates  the  proto- 
plasm of  the  amoeba  but  it  never  develops  there;  it  passes  into 
the  nucleus  through  Ijie  membrane  which  it  perforates,  most  cer- 
tainly through  the  aid  of  a  dissolving  diastase.  Dangeard  has 
demonstrated  the  portal  of  entrance  of  the  parasite  as  a  minute 
circular  opening,  as  though  made  by  a  punch,  persisting  after 
the  entrance  of  the  parasite.  After  its  penetration  into  the  nu- 
cleolus the  parasite  resembles  a  refractile  corpuscle,  increasing 
slowly  in  size  in  proportion  as  the  nuclear  substance  disappears. 
When  this  nuclear  material  has  been  utilized  completely  the  entire 
interior  of  the  nucleus  is  filled  and  the  membrane  is  distended. 
At  this  time  the  nucleus  of  the  parasite,  up  to  the  present  time 
single,  actively  divides  and  when  sporulation  is  effected  there 
are  about  one  hundred  regularly  spaced  nuclei.  About  each  of 
these  nuclei  a  zoospore  organizes,  and  a  sporangium  is  thus  formed, 
containing  distinct,  rounded  corpuscles,  which  contain  nuclei 
at  the  time  of  sporulation. 

Frequently  a  single  amoeba  is  parasitized  by  two  or  perhaps 
several  zoospores,  and  in  such  cases  each  develops  separately  and 
gives  birth  to  a  distinct  sporangium.  When  the  sporangium 
reaches  maturity  the  protoplasm  of  the  amoeba  disintegrates, 
the  sporangium  ruptures,  freeing  the  young  zoospores,  and  these 
become  distributed  throughout  the  medium,  ready  to  parasitize 
the  healthy  amoebae  in  their  neighborhood. 

It  is  evident  that  I  have  not  made  any  comparison  between 
Nucleophaga  amoeba  and  Bacteriophagum  intestinale,  and  that 
these  observations  are  mentioned  simply  because  there  is  a  cer- 
tain resemblance  between  the  two  phenomena  of  destruction, 
that  of  the  amoeba  and  that  of  the  bacterium. 

THE  LYSINS  OF  THE  BACTERIOPHAGE7 

It  is  obvious  that  the  bacteriophage  is  unable,  merely  by  its 
presence,  to  dissolve  a  bacterium.  This  action  can  only  be 
accomplished  through  the  agency  of  lytic  diastases. 

7  The  experiments  dealing  with  the  lysins  have  been  performed  in  col- 
laboration with  G.  Eliava. 


124  THE  BACTEBIOPHAGE 

In  a  culture  of  bacteriophage  the  lysins  which  effect  the  solu- 
tion of  the  bacteria  ought  to  remain  in  solution  when  lysis  is  com- 
pleted. On  the  other  hand,  the  ultramicrobe  does  not  resist 
treatment  with  alcohol.  Therefore,  in  order  to  obtain  lysin  it 
is  only  necessary  to  subject  the  culture  of  bacteriophage  to  the 
classic  procedure  for  the  separation  of  diastases. 

If  we  mix  one  volume  of  bacteriophage  culture  (anti-dysentery) 
with  nine  volumes  of  96  per  cent  alcohol,  after  contact  for  48  hours 
the  precipitate  which  is  formed  is  well  compacted  and  the  super- 
natant fluid  may  be  decanted.  The  precipitate,  which  contains 
the  lysins  admixed  with  all  the  substances  of  the  medium  precipi- 
table  by  alcohol,  is  almost  completely  soluble  in  saline.8 

Experiment  XXXIII.  Precipitate  a  culture  of  anti-dysentery  bacterio- 
phage with  alcohol  and  dissolve  the  precipitate  in  a  quantity  of  0.8  per 
cent  saline  equal  to  the  original  volume  of  the  culture.  Mix  equal  parts 
of  this  solution  and  bouillon  and  add  a  B.  dysenteriae  suspension  sufficient 
to  give  a  slight  turbidity.  As  a  control,  prepare  a  tube  containing  an 
equal  volume  of  bacilli  suspended  in  a  medium  half  bouillon  and  half 
saline.  Place  these  tubes  in  an  incubator  at  37°C. 

After  twenty-four  hours  the  control  is  turbid,  the  bouillon  containing 
the  lysin  is  slightly  cloudy.  Plantings  on  agar  from  the  two  tubes  give 
normal  bacillary  growths. 

After  forty-eight  hours  the  control  presents  the  same  appearance  and 
agar  inoculation  gives  a  perfect  growth.  The  culture  containing  the  lysin 
is  slightly  cloudy  and  inoculations  on  agar  give  only  isolated  colonies.  A 
count  shows  that  there  are  22  times  less  living  bacilli  in  the  last  culture 
than  in  the  control  tube. 

After  three  days  the  appearance  is  the  same  as  after  forty-eight  hours. 

After  four  days  the  bacteria  begin  to  develop  a  resistance  to  the  action 
of  the  lysin,  the  medium  becomes  cloudy  and  inoculations  on  to  agar  again 
give  a  film  of  growth. 

At  no  time  does  one  obtain  on  the  agar  the  plaques  character- 
istic of  the  presence  of  the  bacteriophage,  and  the  action  is  not 
continued  in  series. 

The  alcohol  precipitate  therefore  contaias  a  lytic  diastase, 
free  of  living  ultramicrobes.  The  dissolving  action,  although 
definite,  is  weak;  but  on  the  contrary  as  we  will  see  later, 
the  lysin  manifests  itself  by  an  extremely  powerful  opsonic  action. 

8  This  manipulation  should  be  carried  out  aseptically,  since  filtration 
of  the  fluid  through  a  bougie  considerably  weakens  its  activity. 


BACTERIOPHAGOUS  TJLTRAMICBOBE  125 

OPSONIC  POWER  OF  THE  LYSINS 

In  the  following  experiments  the  opsonic  power  has  been  de- 
termined by  the  method  of  Wright  and  Douglas,  making  a  mix- 
ture of  one  part  of  the  fluid  of  which  the  opsonic  action  is  to  be 
measured,  one  part  of  a  suspension  of  leucocytes,  and  one  part 
of  a  suspension  of  the  bacteria  against  which  the  opsonic  effect 
is  to  be  determined.  The  mixture  is  aspirated  in  a  capillary 
pipette  which  is  sealed  and  placed  in  the  water-bath  at  38°C.  for 
fifteen  minutes.  The  contents  of  the  pipette  are  then  spread  on 
a  slide,  stained,  and  examined. 

As  reagents  we  have  taken:  Guinea  pig  leucocytes,  a  culture 
of  an  anti-Shiga  bacteriophage,  and  a  suspension  of  Shiga  bacilli. 

Experiment  XXXIV 

1.  Control. 

Leucocytes,  bacilli,  ordinary  bouillon 
100  leucocytes  phagocytize  36  bacilli 

Opsonic  index  =    1 

2.  Leucocytes,  bacilli,  culture  of  bacteriophage  two  years  old 
100  leucocytes  phagocytize  692  bacilli 

Opsonic  index  =  19.2 

3.  The  same  mixture,  except  the  bacteriophage  culture  is  diluted  1:250 
100  leucocytes  phagocytize  156  bacilli 

Opsonic  index  =    4.3 

4.  Leucocytes,  bacilli,  culture  of  bacteriophage  six  days  old 
100  leucocytes  phagocytize  1510  bacilli 

Opsonic  index  =  41 . 9 

5.  The  same  mixture,  except  the  bacteriophage  culture  is  diluted  1 :250 
100  leucocytes  phagocytize  146  bacilli 

Opsonic  index  =    4.1 

6.  The  same  mixture,  except  the  bacteriophage  culture  is  heated  at 

60 QC.  for  30  minutes 
100  leucocytes  phagocytize  728  bacilli 

Opsonic  index  =  20.2 

7.  The  same  mixture,  except  the  bacteriophage  culture  is  heated  and 

diluted  to  1 : 250 
100  leucocytes  phagocytize  101  bacilli 

Opsonic  index  =    2.7 

In  the  mixtures  2,  4,  and  6,  the  indices  recorded  represent  a 
minimum.  Many  leucocytes  contain  such  a  number  of  phagocytized  bacilli 
that  counting  is  impossible.  Since  in  the  above  counts  only  those  cells 


126 


THE   BACTERIOPHAGE 


which  did  not  contain  masses  of  bacteria  have  been  included,  the  actual 
index  is  therefore  somewhat  higher.9 

The  opsonic  action  of  a  culture  of  the  bacteriophage  manifests 
itself  with  such  rapidity  that  it  is  improbable  that  the  opsonic 
power  can  be  exercised  directly  by  the  ultramicrobes.  We  have 
seen,  in  fact,  that  the  bacteria  are  parasitized  only  after  an  ap- 
preciable lapse  of  time, — ten  to  twenty  minutes. 

Experiment  XXXV.  Leucocyte  suspension,  Shiga  suspension,  and 
anti-Shiga  bacteriophage  culture  are  mixed  in  equal  parts.  After  various 
periods  of  incubation  drops  of  the  mixture  are  examined  showing : 


TIME  INTEKVAL 

NUMBER  OF  BACILLI 
IN  100  LEUCOCYTES 

INDEX 

Immediately  after  mixing 

197 

5.4 

After  2^  minutes  

362 

10.0 

After  5  minutes 

372 

10.3 

After  7^  minutes  

440 

12.2 

After  10  minutes                            

824 

23.0 

After  ten  minutes  some  of  the  leucocytes  are  so  completely  filled  with 
bacilli  that  counting  is  impossible.  The  figure  given  is  a  minimum  based 
only  on  leucocytes  in  which  masses  of  bacteria  were  not  present  to  interfere 
with  enumeration. 

The  opsonic  power  must  be  exercised,  not  by  the  ultramicrobes, 
but  by  the  lysin  contained  in  the  culture,  as  the  following  ex- 
periment proves. 

Experiment  XXXVI.  Two  milligrams  of  the  alcohol  precipitate  (of 
which  we  have  spoken  above),  still  moist,  are  dissolved  in  10  cc.  of  0.8 
per  cent  saline.  A  mixture  is  made  of  equal  parts  of  this  solution,  sus- 
pension of  Shiga  bacilli,  and  leucocytic  suspension.  After  15  minutes  at 
38°C.  microscopic  examination  of  stained  preparations  shows  that  100 
leucocytes  have  taken  up  536  bacilli  (as  certain  leucocytes  contain  masses 
rendering  a  count  impossible  the  figure  is  a  minimum).  The  index  is  14.9. 

The  opsonic  power  of  cultures  of  the  bacteriophage  is,  there- 
fore, due  to  the  lysin  secreted  by  the  ultramicrobes,  to  the  lysin 
which  remains  in  the  culture  once  the  bacteria  have  been  dis- 

9  It  will  be  recognized  that  the  opsonic  indices  obtained  with  sera  are 
far  below  these  secured  with  cultures  of  the  bacteriophage.  With  the 
former  an  index  of  2  is  exceptional. 


BACTERIOPHAGOUS  ULTBAMICROBE  127 

solved.     It  is  interesting  to  note  its  action  on  bacteria  resistant 
to  the  action  of  the  bacteriophage. 

Experiment  XXXVII.  Mix  equal  parts  of  a  suspension  of  Shiga  bacilli 
resistant  to  the  action  of  the  bacteriophage,  an  anti-Shiga  bacteriophage 
culture,  two  years  old,  and  a  suspension  of  leucocytes.  After  fifteen 
minutes,  100  leucocytes  have  ingested  8  bacteria.  The  index  is  thus  0.22, 
or  90  times  lees  than  with  normal  bacilli  (experiment  XXXIV,  2). 

Prepare  a  similar  mixture,  but  with  a  bacteriophage  culture  six  days  old. 
Here,  100  leucocytes  have  phagocytized  13  bacilli.  The  index  is  0.38,  or 
108  times  less  than  with  normal  bacilli  (experiment  XXXIV,  4). 

Another  mixture  is  made,  using  the  lysin  solution,  100  leucocytes  have 
phagocytized  19  bacilli.  The  index  is  0.53,  or,  28  times  less  than  with 
normal  bacilli  (experiment  XXXVI). 

From  this  it  is  clear  that  bacteria  which  resist  the  bacteriophage 
also  resist  phagocytosis. 

The  same  experiment  has  been  performed  with  a  strain  of  the 
antibarbone  bacteriophage  and  the  bacterium  of  barbone.  The 
results  are  comparable. 

Experiment  XXXVIII   (A)  1.  Control.    Mixture  of  equal  parts  of  leu- 
cocyte suspension,  bouillon,  and  suspension  of  the  bacterium  of  barbone. 
After  fifteen  minutes  at  38°C.  there  are  no  bacteria  in  100  leucocytes. 

2.  Mixture  of  equal  parts  of  leucocyte  suspension,  the  suspension  of  the 
bacterium  of  barbone,  and  an  anti-barbone  bacteriophage  culture,  8  months 
old. 

After  fifteen  minutes  100  leucocytes  have  ingested  109  bacteria. 

3.  The  same  mixture,  except  that  the  bacteriophage  culture  is  diluted 
1 : 250. 

One  hundred  leucocytes  have  phagocytized  52  bacteria. 

4.  Mixture  of  one-third  leucocyte  suspension,  one-third  bacterial  sus- 
pension, and  one-third  solution  of  the  alcohol  precipitate  of  a  recent  culture 
of  the  anti-barbone  bacteriophage  (2  mgm.  of  precipitate  in  10  cc .  of  saline) . 

One  hundred  leucocytes  have  phagocytized  239  bacteria. 

(B)  A  mixture  is  made  of  equal  parts  of  leucocyte  suspension,  culture 
of  the  bacterium  of  barbone,  and  a  fresh  (four  days  old)  culture  of  anti- 
barbone  bacteriophage.  During  incubation  at  38°C.  drops  taken  for 
examination  show: 

Immediately,  in  100  leucocytes  there  are  30  bacteria. 

After  two  and  one-half  minutes,  in  100  leucocytes  there  are  139  bacteria. 

After  five  minutes,  in  100  leucocytes  there  are  201  bacteria. 

After  seven  and  one-half  minutes,  in  100  leucocytes  there  are  271 
bacteria. 

After  ten  minutes,  in  100  leucocytes  there  are  269  bacteria. 

In  the  control  mixture  made  with  bouillon  no  bacteria  were  phago- 
cytized. 


128  THE  BACTERIOPHAGE 

Here  it  is  impossible  to  calculate  the  opsonic  indices,  since  no 
phagocytosis  occurred  in  the  control  mixture.  The  indices  are 
infinity. 

The  bacterium  of  barbone  which  resists  the  action  of  the  bac- 
teriophage  is  also  resistant  to  phagocytosis. 

Experiment  XXXIX.  Prepare  a  mixture  of  one-third  leucocytic  sus- 
pension, one-third  of  the  same  culture  of  anti-barbone  bacteriophage  as 
that  used  in  the  preceding  experiment,  and  one-third  of  a  suspension  of 
the  bacterium  of  barbone  resistant  to  lysis.  After  fifteen  minutes  at  37°C. 
100  leucocytes  have  ingested  3  bacteria,  that  is  to  say,  90  times  less  than 
with  normal  bacteria. 

The  strain  of  anti-dysentery  bacteriophage  employed  in  the 
experiments  previously  described  manifests  a  definite,  although 
feeble,  lytic  action  against  B.  typhosus.  The  following  experi- 
ments show  that  it  also  exerts  a  definite  opsonic  action  on  this 
bacillus. 

Experiment  XL.  1.  Mix  equal  parts  of  bouillon,  leucocyte  suspension, 
and  B.  typhosus  suspension. 

After  fifteen  minutes  at  38°C.  100  leucocytes  have  phagocytized  68 
bacilli.  The  opsonic  index  is  1 . 

2.  Mix  equal  parts  of  anti-Shiga  bacteriophage  culture,  leucocyte  sus- 
pension, and  typhoid  suspension, 

After  fifteen  minutes  100  leucocytes  have  ingested  203  bacilli.  Opsonic 
index  =  3. 

3.  Mix  equal  parts  of  leucocyte  suspension,  typhoid  suspension  and 
lysin  solution  (the  same  one  as  that  employed  in  the  experiments  with  the 
dysentery  bacillus). 

After  fifteen  minutes  100  leucocytes  have  ingested  109  bacilli.  Opsonic 
index  =  1.6. 

The  lysin  possesses  a  property  which  is  indeed  peculiar.  When 
used  in  the  complement  fixation  reaction  as  an  antibody  it  func- 
tions as  an  amboceptor.  The  experiment  cited  below  is  taken 
from  among  many  others  which  gave  identical  results. 

Experiment  XL1.  Antigen:  This  is  prepared  according  to  the  method 
of  Maurice  Nicolle.  One  loopful  of  an  agar  culture  of  B.  dysenteriae  Shiga 
is  suspended  in  4  cc.  of  saline.  This  suspension,  heated  at  100°C.  for  five 
minutes,  then  cooled,  serves  as  antigen. 

Antibody:  An  alcohol  precipitate  of  a  culture  of  anti-Shiga  bacteriophage 
taken  into  solution  in  a  quantity  of  saline  equal  to  the  original  volume  of 
the  culture  acts  as  antibody. 


BACTEKIOPHAGOUS  ULTBAMICROBE 


129 


The  complement  is  fresh  guinea  pig  serum,  titrated. 
The  hemolytic  system  is  the  usual  anti-sheep  system. 


TUBE 

ANTI- 
GEN 

ANTI- 
BODY 

COMPLE- 
MENT 

SALINE 

HEMO- 
LYTIC 

SYSTEM 

RESULT 

CC. 

CC. 

CC. 

CC. 

i  p 

CC. 

1 

0.5 

0.2 

0.2 

1.6 

f  g 

1 

+        + 

2 

0.5 

0.4 

0.2 

1.4 

®  £ 

1 

-J-        -j- 

3 

0.5 

0.5 

0.2 

1.3 

*i  g 

1 

+        +        + 

4 

0.5 

0.6 

0.2 

1.2 

.3  ^ 

1 

+        +        + 

5 

0.5 

— 

0.2 

1.8 

1  1 

1 

Complete  hemolysis 

6 

— 

0.6 

0.2 

1.7 

|| 

1 

Complete  hemolysis,   rapid 

7 

— 

— 

0.2 

2.3 

1 

Complete  hemolysis 

8 

— 

— 

— 

2.5 

J  I 

1 

+     +     +     + 

By  itself,  the  lysin  does  not  fix  complement,  on  the  contrary,  as 
shown  by  tube  6,  it  rather  activates  hemolysis.  There  is,  there- 
fore, an  antibody  associated  with  the  lysin  which  fixes  the  com- 
plement. 

It  is  difficult  to  reach  a  conclusion  regarding  this  curious  ex- 
periment. Later  investigations  will  show  that  there  is  a  relation 
between  the  amboceptor  of  Bordet  and  the  lysin  of  the 
bacteriophage,  since  it  acts  as  two  different  principles,  acting  in 
an  identical  manner,  in  so  far  as  complement  fixation  is  concerned. 

All  of  these  experiments  show  that  the  bacteriophagous  ultra- 
microbe  secretes  a  principle,  precipitable  by  alcohol,  resisting  a 
temperature  of  58°C.,  and  persisting  for  several  months  in  the 
cultures  of  the  bacteriophage.  This  principle,  aside  from  its 
solvent  action,  exercises  a  powerful  opsonic  action  upon  the  bac- 
teria for  which  the  ultramicrobe  from  which  it  is  derived  possesses 
a  virulence.  The  opsonic  activity  appears  proportional  to  the 
virulence  of  the  bacteriophage  for  the  bacterium  under  consid- 
eration. Bacteria  which  have  acquired  a  resistance  to  the  bac- 
teriophage are  equally  resistant  to  phagocytosis.  In  addition, 
from  another  viewpoint,  we  will  see  that  they  possess  an  increased 
virulence. 


CHAPTER   V 

THE  BACTERIOPHAGOUS  ANTISERUMI 

Complexity  of  the  Antibodies.  Antibodies  to  the  Bacteria.  Antibodies 
to  the  Bacterial  Toxins.  Antibodies  to  the  Bacteriophagous  Ultrami- 
crobes.  Antibodies  to  the  Lysins.  Incidental  Conditions  Resulting  from 
the  Existence  of  the  Bacteriophage. 

COMPLEXITY  OF  THE  ANTIBODIES 

The  phenomena  here  involved  are  exceedingly  complex.  It 
is  known  that  when  the  body  is  injected  with  a  bacterial  culture 
it  responds  with  the  production  of  diverse  principles  which  are 
grouped  under  the  name  "antibodies."  Some  of  these  act  upon 
the  bacterial  bodies:  the  agglutinins,  amboceptors,  opsonins; 
others,  the  antitoxins  and  antiferments,  neutralize  the  secretory 
products  of  the  bacteria  formed  in  the  culture  fluid  injected. 
When  a  mixture  of  two  bacterial  cultures  is  injected,  the  body 
responds  with  a  duplicate  series  of  antibodies.  This  takes  place 
when  a  culture  of  the  bacteriophage  is  injected. 

A  culture  of  the  bacteriophage  is  composed,  as  we  know,  of  a 
culture  or  a  suspension  of  a  bacterium  lysed  by  the  action  of  the 
bacteriophage  directed  against  and  endowed  with  virulence  for 
this  bacterium.  The  bacteriophagous  germs  inoculated  have 
multiplied  at  the  expense  of  the  bacterial  bodies  found  there 
and  when  lysis  is  terminated  the  bacterial  substance  is  dissolved 
in  the  medium.  A  culture  of  the  bacteriophage  is,  then,  a  com- 
plex medium  which  contains: 

a.  The  substance  of  the  bacterial  bodies  in  a  dissolved  state. 

6.  The  bacterial  toxins  (exo-  or  endotoxins). 

c.  The  bacteriophagous  ultramicrobes  which  have  developed 
at  the  expense  of  the  bacteria. 

1  The  experiments  performed  on  the  bacteriophagous  antiserum  have 
been  made  in  collaboration  with  G.  Eliava. 

130 


BACTERIOPHAGOUS  ANTISERUM  131 

d.  The  products  resulting  from  the  activity  of  the  bacterio- 
phage,  which  we  have  grouped  under  the  name  of  "lysins,"  and 
which  remain  in  the  medium  after  the  lytic  process  is  completed. 

Does  the  bacteriophage  attack  the  bacterium  by  means  of  a 
single  diastase  or  through  a  combination  of  diastases?  At  this 
time  it  is  impossible  to  say,  and  indeed,  it  would  not  materially 
affect  the  question  with  which  we  are  concerned. 

There  is  still  another  category  of  substances  present  in  the 
culture.  We  have  seen  that  the  bacteria  do  not  remain  passive 
to  the  action  of  the  bacteriophage,  and  that  this  defense  is  ac- 
companied by  the  production  of  an  anti-diastase — an  anti-lysin — 
which  is  likewise  to  be  found  in  the  medium.  This  should,  then, 
stimulate  the  formation  of  anti-anti-lysins.  These  have  not 
been  investigated,  and  it  is  only  sugested  that  they  may  possibly 
be  present  in  the  serum  of  immunized  animals. 

As  an  example  of  an  antibacteriophagous  serum  we  will  take 
the  antibacteriophage-Shiga  serum.  This  is  particularly  interest- 
ing because  of  the  potent  endotoxin  of  the  dysentery  bacillus. 

A  rabbit  is  injected  with  four  doses  of  a  culture  of  the  anti- 
dysentery  bacteriophage2  that  is  to  say,  of  a  lysed  culture  of  B. 
dysenteriae  Shiga,  amounting  to  two,  four,  six,  and  eight  cubic 
centimeters,  with  an  interval  of  six  days  between  each  injection. 
The  rabbit  is  bled  fifteen  days  after  the  last  injection.  Theoreti- 
cally, the  serum  of  this  rabbit  ought  to  contain  the  following  anti- 
bodies: 

a.  Antibodies  to  the  bacteria:  Amboceptor  and  agglutinin. 

b.  Antibody  to  the  bacterial  toxin:  Antitoxin. 

c.  Antibodies    to    the    bacteriophagous    ultramicrobe :  Ambo- 
ceptor and  agglutinin. 

d.  Antibody  to  the  lytic  diastase  of  the  bacteriophage:  Anti- 
lysin. 

Let  us  see  if  the  antibodies  present  in  such  a  serum  actually 
correspond  with  those  which  theoretically  should  exist. 

2  As  we  will  see  in  regard  to  barbone  of  the  buffalo,  the  serum  of  an  animal 
which  has  received  a  single  and  minimal  injection  of  a  bacteriophage  culture 
does  not  present  the  antibacteriophagous  property,  or,  at  least,  if  it  exists, 
it  is  not  detectable. 


132  THE  BACTERIOPHAGE 


ANTIBODIES  TO  THE  BACTERIA 

The  dysentery  bacilli  are  agglutinated  by  the  antibacterio- 
phagous  serum,  and  this  serum  contains  also  an  amboceptor 
which  permits  the  fixation  of  complement.  The  presence  of 
such  antibodies  is  inevitable  and  is  obtained  by  the  injection  of 
any  material  containing  the  dysentery  bacilli,  living  or  dead, 
intact  or  dissolved.  The  presence  of  such  antibodies  is  without 
especial  significance. 

ANTIBODIES  TO  THE  BACTERIAL  TOXIN 

In  the  present  case  these  antibodies  should  neutralize  the  dysen- 
tery endotoxin.  The  serum  of  an  animal  prepared  by  the  in- 
jection of  dysentery  bacilli,  living  or  dead,  contains  an  antitoxin.3 
On  the  other  hand,  a  culture  of  B.  dysenteriae  lysed  by  the  bac- 
teriophage  contains  a  toxin,  for  if  experimental  animals  are  in- 
jected with  such  a  culture  a  short  time  after  lysis  the  animals  die 
as  though  they  had  received  a  lethal  dose  of  the  toxin  of  Nicolle. 
The  serum  of  an  animal  treated  with  such  cultures  ought  to  con- 
tain an  antitoxin.  This  can  be  verified. 

Experiment  XL1I.  A  mouse  receives  by  subcutaneous  injection  a  lethal 
dose  of  the  dysentery  toxin  prepared  by  the  method  of  Nicolle,  and  at  the 
same  time  0.5  cc.  of  the  bacteriophage-Shiga  antiserum.  A  second  mouse 
receives  the  same  amount  of  toxin  and  0.5  cc.  of  an  anti-dysentery  serum. 
A  third  mouse  receives  a  lethal  dose  of  the  toxin  only.  The  first  mouse 
dies  in  about  thirty  hours  after  the  injection,  the  second  lives,  the  third 
dies  four  days  after  the  injection. 

The  bacteriophage-Shiga  antiserum  is  therefore  not  antitoxic; 
indeed,  on  the  contrary,  it  is  definitely  sensitizing.  Let  us  con- 
sider this  singular  phenomenon  further. 

3  The  antidysenteric  serum  furnished  by  the  Pasteur  Institute  is  derived 
from  horses  treated  by  injections  of  dysentery  toxin  secured  according 
to  the  method  of  Rowland,  as  modified  by  Maurice  Nicolle.  The  bacterial 
bodies  are  ground  with  anhydrous  sodium  sulfate,  the  powder  obtained  is 
dried  in  the  air,  and  dissolved  in  water  at  the  time  of  injection.  The 
turbid  fluid  thus  obtained  is  centrifuged,  and  the  clear  supernatant  portion 
is  used  for  the  injection.  The  serum  neutralizes  the  endotoxin,  as  animal 
experimentation  shows. 


BACTERIOPHAGOUS  ANTISERUM  133 

Experiment  XLIII.  Four  mice  receive  subcutaneously  a  dose  of  toxin 
equal  to  one-tenth  of  the  lethal  dose.  The  first  is  held  as  a  control.  Two 
others  receive  0.2  cc.  of  the  bacteriophage-Shiga  antiserum,  the  last  0.1 
cc.  of  this  serum.  The  first  remains  perfectly  well  indefinitely.  The  two 
which  received  the  0. 2  cc.  dose  of  serum  die  after  40  hours,  and  the  last  one 
after  fifty-four  hours. 

This  experiment  proves  that  the  bacteriophage-Shiga  anti- 
serum  sensitizes  the  animal  to  the  action  of  the  toxin.  It  should 
be  stated  here  that  whatever  the  number  of  lethal  doses  of  the 
toxin  of  Nicolle  injected  into  a  mouse,  death  never  occurs  before 
the  fourth  day.  Here,  when  the  antibacteriophage  serum  is 
added  to  the  toxin,  even  to  a  dose  below  the  minimal  lethal  dose, 
death  takes  place  within  forty-eight  hours. 

Instead  of  toxin,  let  us  take  living  dysentery  bacilli  and  see  the 
effect  of  the  antiserum  on  injections  of  this  nature. 

Experiment  XLIV .  Four  mice  receive  subcutaneously  a  dose  of  dysen- 
tery bacilli  equal  to  one-fifth  the  lethal  dose.  The  first  mouse  is  held  as  a 
control.  The  second  receives,  subcutaneously,  0. 2  cc.  of  the  antibacterio- 
phage-Shiga  serum,  the  last  two  0. 1  cc.  of  this  serum.  The  control  animal 
lives,  showing  nothing  abnormal.  Those  which  at  the  same  time  received 
the  serum  die  in  seven  to  nine  days  after  the  injection,  after  showing  during 
the  last  twenty-four  hours  a  paralysis  of  the  posterior  extremities.  In 
general,  mice  do  not  show  this  symptom  after  the  injection  of  B.  dysen- 
teriae;  only  the  rabbit  shows  this  particular  symptom. 

The  result  is  clear-cut;  in  all  cases  the  antibacteriophage-Shiga 
serum  is  sensitizing.  This  is,  incidentally,  the  first  example  of 
an  anti-immunizing  serum. 

It  is  possible  that  the  antibacteriophage-Shiga  serum  actually 
contains  an  antitoxin  but  that  this  is  masked  by  the  presence  of 
a  powerful  "sensibilisine."  This  is  the  more  plausible,  for  we 
shall  see  in  the  chapter  dealing  with  immunization,  that  rabbits 
which  have  received  but  a  single  minute  injection  of  a  culture  of 
the  antidysentery  bacteriophage  are  effectively  vaccinated  against 
the  effects  of  the  toxin.  The  sensibilisine  develops  in  the  animal 
only  after  the  second  injection.  This  condition  is  not  peculiar 
to  the  case  of  dysentery ;  we  find  it  again  when  we  consider  immuni- 
zation against  barbone. 

This  phenomenon  of  sensitization  invites  much  further  investi- 
gation, which  will  permit,  without  doubt,  an  extension  of  our 


134  THE  BACTEKIOPHAGE 

knowledge  of  the  nature  of  antitoxic  immunity.  One  thing  already 
appears  certain,  namely,  that  the  bacteriophage  must  play  some 
r61e  in  the  manifestation  of  this  immunity,  since  the  antibacterio- 
phagous  serum  sensitizes  to  the  toxins.  It  is  probable  that  this 
sensitization  of  the  animal  must  be  associated  with  the  inhibiting 
power  with  which  the  antibacteriophage  serum  is  endowed,  an 
action  which  we  will  shortly  consider. 

ANTIBODIES   TO   THE    BACTERIOPHAGOTJS   ULTRAMICROBES 

Agglutinins 

It  has  been  impossible  to  demonstrate  definitely  the  presence 
of  agglutinins,  although  experimentation  indicates  that  their 
presence  is  probable.  In  all  cases,  if  present,  they  are  weakly 
active. 

Experiment  XLV.  When  a  culture  of  the  bacteriophage  is  centri- 
fuged  for  15  minutes  at  3000  revolutions  per  minute  no  trace  of  sediment 
appears;  it  requires  a  speed  of  at  least  12,000  revolutions  to  obtain  an 
appreciable  amount .  A  culture  of  the  bacteriophage  is  mixed  with  one-tenth 
of  its  volume  of  antibacteriophage  serum  and  centrifuged  for  ten  minutes 
at  3000  revolutions.  Counts  of  the  ultramicrobes  in  the  sediment  and  in 
the  supernatant  fluid  (after  energetic  shaking  of  the  latter  for  five  minutes) 
shows  that  there  are  about  three  times  as  many  germs  in  the  sediment  as 
in  the  supernatant  fluid. 

Is  this  agglutination?  It  is  possible,  but  not  certain.  The 
experiment  is  not  sufficiently  clear-cut  to  permit  an  affirmative 
answer.  The  formation  of  agglutinins  in  the  serum  of  treated 
animals  is  subject  to  great  variation,  dependent  upon  the  bac- 
terial species  injected.  With  Vibrio  cholerae  and  with  B.  typhosus 
for  example,  potent  agglutinins  are  secured;  with  certain  coli- 
form  bacilli  and  with  B.  Friedldnder  they  are  usually  so  weak  that 
their  action  is  almost  inappreciable.  We  know  nothing  regard- 
ing the  formation  of  agglutinins  in  the  case  of  the  pathogenic 
ultramicrobes. 

Amboceptors 

The  test  for  an  amboceptor  specific  for  the  anti-dysenteric 
bacteriophage,  detectable  by  the  complement  fixation  reaction 


BACTERIOPHAGOUS  ANTISEBUM  135 

of  Bordet  and  Gengou,  can  not  be  made.  In  effect,  the  culture 
of  the  anti-dysentery  bacteriophage  is  only  a  suspension  of  ul- 
tramicrobes  in  a  liquid  containing  the  dissolved  substance  of  the 
dysentery  bacilli,  so  that  the  antibacteriophage-Shiga  serum  con- 
tains two  amboceptors,  one  specific  for  the  dissolved  substance, 
the  other  for  the  ultramicrobe,  and  it  is  impossible  to  separate 
the  two  actions.  A  fixation  of  complement  would  always  be 
obtained  without  the  possibility  of  knowing  with  which  of  the 
two  antigens  it  had  been  effected.  However,  the  question  can 
be  solved  in  another  manner;  a  manner  which  is  very  conclusive. 

We  have  considered  in  the  preceding  chapters  the  experiments 
which  demonstrate  the  unicity  of  the  bacteriophage.  If  the 
hypothesis  based  on  these  experiments  is  true  the  amboceptor 
present  in  the  antibacteriophage-Shiga  serum  ought  to  fix  comple- 
ment with  all  ultramicrobes  and  the  detection  of  an  amboceptor 
ought  to  be  possible,  for  working  with  a  culture  of  bacteriophage 
other  than  the  antidysenteric  there  would  be  nothing  to  inter- 
fere with  the  reaction.  A  culture  of  antiplague  bacteriophage, 
for  example,  is  a  suspension  of  ultramicrobes  in  a  fluid  containing 
the  substance  of  B.  pestis.  The  only  possible  common  element 
in  a  culture  of  antidysentery  bacteriophage  and  in  a  culture 
of  antiplague  bacteriophage  is  the  bacteriophagous  ultramicrobe. 
And  the  single  "anti"  element  contained  in  an  antibacterio- 
phage-Shiga serum  capable  of  exercising  an  action  toward  such  a 
plague  culture  can  only  be  an  amboceptor  for  the  single  element 
common  to  all  cultures  of  the  bacteriophage;  the  bacteriophagous 
ultramicrobes  themselves. 

The  complement  fixation  reaction  has  been  conducted  utiliz- 
ing as  antibody  the  antibacteriophagous-Shiga  serum;  as  anti- 
gens, cultures  of  bacteriophage  for  dysentery,  plague  of  human 
origin,  an  anti-plague  strain  from  rats,  and  an  anti-barbone 
strain  from  the  buffalo. 

Experiment  XLVI.    Fixation  of  complement. 

Antigen:  culture  of  anti-Shiga  bacteriophage  containing  2,000,000,000 
ultramicrobes  per  cubic  centimeter. 

Antibody:  antibacteriophage-Shiga  serum. 

With  the  antigen  in  an  amount  of  1  cc.  and  antibody  in  quantities  of  0.2 
and  0.1  cc.  fixation  is  positive. 


136 


THE   BACTEKIOPHAGE 


Moreover,  the  antigen  by  itself  fixed  complement. 

Experiment  XLVII.  Antigen :  culture  of  anti-Shiga  bacteriophage  con- 
taining 100,000,000  ultramicrobes  per  cubic  centimeter. 

Antibody:  antibacteriophage-Shiga  serum. 

Positive  fixation  was  secured  with  mixtures  containing  the  antigen  in 
1  cc.  amounts  and  the  serum  in  0. 2  and  0.1  cc.  quantities. 

The  antigen  by  itself  did  not  fix  complement. 

Experiment  XLVIII.    Antigen:  B.  dysenteriae  Shiga. 

Antibody:  antibacteriophage-Shiga  serum. 

Fixation  of  complement  occurred. 

Experiment  XLIX.    Antigen:  culture  of  the  anti-Shiga  bacteriophage. 

Antibody:  an  antidysentery  serum. 

Complement  is  fixed. 

Experiment  L.    Antigen:  culture  of  the  anti-Shiga  bacteriophage. 

Antibody:  antibacteriophage-Shiga  serum. 

The  antigen,  antibody,  and  complement  are  incubated  together  for  1 
hour  at  37°C.,  and  then  the  hemolytic  system  is  added. 

The  following  protocol  shows  the  results. 


TUBE 

ANTI- 
GEN 

ANTI- 
BODY 

COM- 
PLE- 
MENT 

1:20 

SALINE 

HEMO- 
LYTIC 
SYSTEM 

RESULT 

CC. 

CC. 

cc. 

CC. 

CC. 

1 

0.75 

0.2 

0.2 

0.35 

1 

+        +        +        + 

2 

0.50 

0.2 

0.2 

0.60 

1 

+     +     +     +  (optimum) 

3 

0.25 

0.2 

0.2 

0.85 

1 

+     +     + 

4 

0.75 

0.1 

0.2 

0.45 

1 

+     +     +     + 

5 

0.75 

0.3 

0.2 

0.25 

1 

+     +     +     + 

6 

0.75 

— 

0.2 

0.55 

1 

Complete  hemolysis 

7 

0.75 

0.2 

0.3 

0.25 

1 

+     +     +     (excess  complement) 

8 

0.75 

0.2 

0.4 

0.15 

1 

-)-     +      (excess    complement) 

9 

— 

0.3 

0.2 

1.00 

1 

Complete  hemolysis 

Experiment  LI.    Antibody:  antibacteriophage-Shiga  serum. 
Antigens:  Four  different  antigens  are  employed,  as  follows: 

I.  Anti-Shiga  bacteriophage,  1,500,000,000  ultramicrobes  per  cubic 

centimeter. 

II.  Anti-barbone  bacteriophage,  250,000,000  ultramicrobes  per  cubic 
centimeter. 

III.  Anti-plague    bacteriophage,    a    strain    derived    from    the    rat, 

450,000,000  ultramicrobes  per  cubic  centimeter. 

IV.  Anti-plague  bacteriophage,  a  strain  derived  from  a  case  of  plague 

in  man,  700,000,000  ultramicrobes  per  cubic  centimeter. 
The  results  secured  with  these  four  different  antigens  are  shown  in  the 
following  table. 


BACTERIOPHAGOUS  ANTISERUM 


137 


ANTI- 
GEN 

ANTI- 
BODY 

NORMAL 
RABBIT 

SERUM 

COM- 
PLE- 
MENT 

1:20 

SALINE 

HEMO- 
LYTIC 

SYSTEM 

ANTIGENS 

I 

II 

ill 

IV 

CC. 

CC. 

CC. 

CC. 

CC. 

CC. 

1 

0.2 

— 

0.2 

1.1 

1 

+  +  +  + 

+++ 

++++ 

++++ 

1 

0.1 

— 

0.2 

1.2 

1 

+  +  +  + 

-}--f 

+-H+ 

++++ 

— 

0.2 

— 

0.2 

2.1 

1 

CH 

CH 

CH 

CH 

1 

— 

— 

0.2 

1.3 

1 

CH 

CH 

CH 

CH 

— 

— 

— 

0.2 

2.3 

1 

CH 

CH 

CH 

CH 

1 

— 

0.5 

0.2 

1.3 

1 

+ 

+ 

+ 

+ 

=  Complete  inhibition          CH  =  Complete  hemolysis 

Experiment  LII.    The  antibody  and  the  antigens  are  the  same  as  those 
used  in  the  preceding  experiment .    The  following  table  presents  the  results. 


ANTI- 
GEN 

ANTI- 
BODY 

NORMAL 
RABBIT 
SERUM 

COM- 
PLE- 
MENT 

1:20 

SALINE 

HEMO- 
LYTIC 
SYSTEM 

ANTIGENS 

I 

II 

ill 

IV 

CC. 

CC. 

CC. 

CC. 

CC. 

CC. 

1 

0.05 

— 

0.2 

1.25 

1 

+  +  +  + 

+  +  + 

+  +  +  + 

++++ 

1 

0.025 

— 

0.2 

.30 

1 

+  +  +  + 

_|__j_ 

_!__{_ 

++++ 

1 

0.005 

— 

0.2 

.30 

1 

-f-f-f 

C  H 

C  H 

CH 

1 

— 

0.05 

0.2 

.25 

1 

C  H 

C  H 

C  H 

C  H 

1 

— 

0.025 

0.2 

.30 

1 

C  H 

C  H 

C  H 

C  H 

1 

— 

0.005 

0.2 

.30 

1 

C  H 

C  H 

C  H 

C  H 

- 

0.05 

- 

0.2 

2.25 

1 

Complete  hemolysis 

— 

— 

0.05 

0.2 

2.25 

1 

Complete  hemolysis 

1 

— 

— 

0.2 

1.30 

1 

C  H 

C  H 

C  H 

C  H 

— 

- 

- 

0.2 

2.30 

1 

Complete  hemolysis 

~ 

~ 

mm 

— 

2.50 

1 

No  hemolysis 

It  will  be  noted  (experiment  LI)  that  the  antigen  slightly  fixed 
complement  with  normal  rabbit  serum.  This  is  not  strange  since 
the  bacteriophage  is  a  normal  inhabitant  of  the  intestine. 

The  antibacteriophage  serum  contains,  therefore,  an  amboceptor 
specific  for  the  bacteriophage,  whatever  the  strain  may  be,  what- 
ever the  species  of  bacteria  attacked,  and  whatever  the  animal 
species  from  which  it  is  derived.  There  is  but  one  bacteriophage. 


138  THE   BACTERIOPHAGE 

ANTIBODIES  TO  THE  LYSINS4 

We  have  already  seen  that  it  is  possible  to  obtain  lysins  without 
admixture  with  viable  bacteriophagous  ultramicrobes  by  pre- 
cipitating a  culture  of  the  bacteriophage  with  alcohol. 

Since  growth  of  the  bacteriophage  takes  place  within  the  in- 
terior of  the  bacteria  the  ultramicrobe  can  effect  its  penetration 
only  by  corroding  the  wall  of  the  bacterium  in  order  to  make  way 
for  its  passage,  and  it  is  evident  that  it  can  do  this  only  by  means 
of  a  lysin.  If  the  antibacteriophage  serum  contains  an  antilysin 
it  is  evident  that  the  penetration  will  be  retarded  or  even  pre- 
vented by  the  presence  in  the  medium  of  the  neutralizing  serum. 
And  this  delay  or  prevention,  according  to  the  amount  and  po- 
tency of  the  serum,  will  be  associated  with  a  delay  or  prevention 
of  the  lysis,  since  the  ultramicrobes  will  be  unable  to  penetrate 
the  bacterial  cells.  The  antibacteriophage  serum  will  assure,  in 
a  word,  the  protection  of  the  bacteria,  without  actually  exercis- 
ing any  action  on  the  vitality  of  the  virus  itself.  This  is,  in  fact, 
what  is  actually  observed. 

The  antibacteriophage  serum  used  in  these  experiments  pos- 
sessed a  considerable  inhibiting  power;  0.00001  cc.  added  to  10  cc. 
of  a  suspension  of  dysentery  bacilli — this  is  one-millionth  of  the 
final  volume — markedly  retarded  lysis.  With  0.001  cc.  lysis 
was  prevented.  Obviously,  this  fact  might  be  interpreted  as  a 
destruction  of  the  bacteriophage  pure  and  simple.  But  this 
would  be  indeed  strange  in  view  of  the  fact  that  a  serum  has  never 
destroyed  a  bacterium  in  vitro,  even  in  the  presence  of  comple- 
ment. I  know  that  this  affirmation  is  contrary  to  universal 
opinion  touching  the  bacteriolytic  action  of  antisera,  but  in 
Part  II  of  this  work  I  will  show  the  foundation  for  it  by  an  ex- 
periment which  permits  of  no  doubt. 

In  so  far  as  the  action  of  an  antibacteriophage  serum  upon 
lysis  is  concerned,  the  following  experiment  shows  that  the  bac- 
teriophage is  not  destroyed;  its  action  is  only  inhibited. 

4  To  repeat  what  is  meant  by  lysin :  The  aggregate  of  the  secretions  of 
the  bacteriophage,  without  prejudging  that  they  operate  as  a  diastase  only, 
or  as  a  collection  of  diastases,  as  is  more  probable.  It  has  been  shown  in 
an  earlier  chapter  that  the  bacteria,  like  higher  organisms,  react  to  the 
lysins  by  the  production  of  antilysins. 


BACTERIOPHAGOUS  ANTISERUM  139 

Experiment  LIII.  Prepare  a  mixture  of  equal  parts  of  antibacterio- 
phage-Shiga  serum  and  culture  of  anti-Shiga  bacteriophage.  Allow  them 
to  remain  in  contact  for  five  days.  They  are  placed  under  conditions  such 
that  if  the  serum  exerted  a  destructive  action  on  the  ultramicrobes  its 
effect  could  not  fail  of  manifestation .  After  these  five  days  of  contact,  three 
tubes,  each  containing  10  cc.  of  sterile  bouillon,  are  seeded  with  a  drop  of 
a  bouillon  culture  of  Shiga  bacilli.  To  the  first  of  these  tubes  add  a  drop  of 
the  mixture  of  &ntiserum-bacteriophage;  to  the  second  add  a  drop  from 
this  first  tube  after  it  has  been  shaken ;  and  to  the  third  add  a  drop  from  the 
second.  We  have  them  a  series  of  three  tubes,  planted  with  B.  dysenteriae 
Shiga,  containing  decreasing  concentrations  of  the  serum-bacteriophage 
mixture.  After  twenty-four  hours  at  37°C.  normal  cultures  of  Shiga  are 
obtained  in  the  three  tubes,  and  plantings  on  agar  likewise  give  normal 
cultures.  Up  to  this  point  it  looks  as  though  the  lytic  principle  has  been 
destroyed. 

Continue  the  experiment.  Replace  the  three  tubes  in  the  incubator  and 
twenty-four  hours  later  it  is  seen  that  lysis  has  commenced  in  the  first 
tube  of  the  series.  Agar  inoculation  from  this  tube  remains  sterile.  The 
last  two,  on  the  contrary,  still  contain  a  normal  culture  of  B.  dysenteriae. 
Return  the  tubes  again  to  the  incubator.  After  twenty-four  hours,  that 
is,  three  days  after  the  beginning  of  the  experiment,  lysis  takes  place  in 
the  last  two  tubes.  All  subcultures  on  agar  remain  sterile. 

The  bacteriophage  is  therefore  not  destroyed  by  the  antibac- 
teriophage  serum;  its  power  is  simply  inhibited  for  a  time. 

The  experiment  further  shows  that  the  action  is  truly  inhibi- 
tive,  acting  upon  the  entire  number  of  ultramicrobes.  In  other 
words,  the  delayed  lysis  is  not  due  to  the  revival  of  certain  ultra- 
microbes  particularly  resistant,  since  lysis  is  produced  even  in 
the  last  tube  which  has  received,  as  a  result  of  successive  dilu- 
tion, an  infinitesimal  quantity  of  the  germs. 

The  presence  of  an  antilysin  in  the  antibacteriophage  serum 
allows  us  to  obtain  further  information  regarding  the  nature  of 
the  virulence  of  the  ultramicrobe. 

Experiment  LIV.  To  a  suspension  of  the  bacterium  of  barbone  add  a 
drop  of  a  bacteriophage  culture  active  against  this  organism  and  then 
10  drops  of  an  antibacteriophage-Shiga  serum,  that  is,  a  quantity  of  serum 
completely  inhibitive  of  lysis  in  a  suspension  of  Shiga  bacilli.  Prepare 
also  a  control  without  the  serum.  In  both  tubes  lysis  proceeds  normally 
and  in  a  parallel  fashion.  Here,  then,  the  serum  has  exerted  no  inhibitory 
action. 


140  THE   BACTERIOPHAGE 

The  inhibitive  effect  is  exercised  only  against  the  strain  of 
bacteriophage  which  has  been  used  to  inject  the  animal  in  the 
preparation  of  the  antiserum.5  On  the  other  hand  we  know  that 
strains  of  the  bacteriophage  differ  one  from  the  other  only  in 
the  virulence  which  they  have  acquired,  by  adaptation,  for  this 
or  that  bacterium.  It  follows  that  the  lysin  secreted  by  the  bac- 
teriophage is  different  for  the  different  bacteria  attacked,  since 
the  antilysin  neutralized  only  the  lysin  of  the  strain  which  has 
served  in  the  treatment  of  the  animal  furnishing  the  serum. 
Each  bacterial  species  requires  for  its  lysis,  then,  the  production 
of  a  specific  lysin. 

Virulence  for  a  given  bacterium  is,  therefore,  in  the  last  analy- 
sis, the  power  possessed  by  the  bacteriophage  to  secrete  a  lysin 
specific  for  this  bacterium. 

The  bacterial  species  are  divided  into  groups.  In  each  group 
the  species  which  compose  it  present  certain  common  character- 
istics. For  example,  we  have  the  colon  group,  the  typhoid  group 
(B.  typhosus  and  the  paratyphoid  bacilli),  the  dysentery  group 
(the  Shiga,  Flexner,  and  Hiss  types,  etc.).  These  three  groups 
are  indeed  closely  related  one  to  another.  On  the  other  hand, 
we  see  the  Pasteurella  group  (bacteria  of  the  diverse  hemorrhagic 
septicemias,  chicken  cholera,  barbone,  etc.),  the  staphylococcus 
group,  and  so  on.  Each  bacterial  species  requires  that  it  be 
attacked  through  the  secretion  of  a  specific  lysin  and  the  differ- 
ence between  these  specific  lysins  will  be  slight  in  passing  from 
one  bacterial  species  to  another  in  the  same  group  or  in  a  neigh- 
boring group.  The  bacteriophage  adapts  itself  rapidly.  A 
single  strain  is  in  fact  generally  active  for  all  the  bacteria  of  the 
group,  or  for  the  organisms  of  the  most  closely  related  ones. 
On  the  contrary,  adaptation  is  difficult  of  acquisition  in  passing 
from  a  bacterium  of  one  group  to  an  organism  of  a  remotely 
related  group. 

Furthermore,  the  bacteriophage  normally  parasitizes  the 
intestinal  bacteria  which  constitute  its  habitual  culture  medium. 

6  It  is  evident  that  if  the  antibacteriophage-Shiga  serum  is  tested  against 
closely  related  bacterial  types,  forms  for  which  the  bacteriophage  would 
have  a  certain  activity,  an  inhibitive  effect  more  or  less  pronounced  will 
be  noted. 


BACTERIOPHAGOUS  ANTISEBUM  141 

It  retains  for  a  long  time  its  hereditary  faculty  of  attacking  the 
organisms  of  the  colon-typhoid-dysentery  group,  even  if  it  is 
cultivated  for  many  generations  at  the  expense  of  another  bac- 
terial species. 

INCIDENTAL     CONDITIONS     RESULTING     FROM     THE     EXISTENCE     OF 
THE  BACTERIOPHAGE 

I  wish  to  note  certain  incidental  consequences  which  spring 
from  the  facts  which  we  have  considered.  Although  accessory, 
these  consequences  are  of  some  practical  significance  and  it  may 
be  well  to  mention  them. 

Because  of  the  ubiquity  of  the  bacteriophage  and  its  con- 
stant presence  in  all  living  beings,  and  because  of  the  resistance 
which  the  bacteria  are  able  to  oppose  to  its  action,  and  which  gives 
birth  to  the  phenomenon  of  mixed  cultures,  it  is  henceforth 
necessary  to  verify  the  purity  of  bacterial  cultures  from  the 
point  of  view  of  possible  contamination  not  only  by  another 
bacterial  species,  but  also  by  an  ultramicrobe. 

We  will  see,  for  example,  that  in  B.  coli  pyelonephritis,  the 
pathogenic  agent  is  always  a  colon  bacillus  which  is  resistant  to 
the  bacteriophage.  If  one  plants  the  urine  from  a  case  of  pyelone- 
phritis on  agar  for  the  purpose  of  isolating  the  etiological  agent 
one  is  always  liable  to  find  mixed  colonies  of  the  colon  bacillus 
and  the  bacteriophage.  Subculture  from  these  mixed  colonies 
will  give  mixed  cultures,  indefinitely  cultivable  in  this  form. 
Such  cultures  are  usually  considered  pure,  for  they  contain  no 
other  organism  visible  under  the  microscope.  They  are,  how- 
ever, contaminated  by  the  bacteriophagous  ultramicrobe;  they 
are  not  "ultrapure,"  and  investigations  undertaken  with  such 
mixed  colon-bacteriophage  cultures  may  furnish  peculiar  results, 
especially  if  they  are  used  in  immunological  experimentation. 
It  is  not  to  be  assumed  that  I  have  mentioned  an  exceptional 
case,  far  from  it,  as  the  two  following  examples  demonstrate. 

In  two  different  instances,  both  accidental  findings,  I  have 
demonstrated  that  cultures  of  the  colon  bacillus  isolated  origi- 
nally from  cases  of  cystitis  in  the  hospital,  were  in  reality  mixed 
cultures.  In  both  instances  it  was  easy  to  isolate  from  these 
cultures  a  very  active  bacteriophage. 


142  THE  BACTERIOPHAGE 

Here  is  another  instance  of  the  same  order.  Recently  investi- 
gating a  bacteriophage  active  for  the  streptococcus  of  gourme  of 
horses,  Doctor  Forgeot  sent  me  three  different  strains  of  Strep- 
tococcus equi,  taken  from  the  culture  collection  of  the  Central 
Veterinary  Laboratory.  Of  these  three  strains,  only  one  was 
pure.  The  two  others  were  in  reality  mixed  cultures  of  the 
streptococcus  and  the  bacteriophage. 

These  two  examples  suffice  to  give  an  idea  of  the  great  num- 
ber of  mixed  cultures  which  must  actually  exist  among  stock  cul- 
tures. Not  only  are  the  intestinal  bacteria  subject  to  contamina- 
tion by  the  bacteriophage,  but  bacteria  in  general,  for  we  will  see 
that  the  bacteriophage  does  not  remain  restricted  to  the  intestinal 
tract,  but  passes  into  the  circulation  and  exercises  its  action  in 
the  different  organs. 

It  is  therefore  quite  essential  before  undertaking  any  experi- 
mental work  to  ascertain  if  the  bacterial  culture  involved  is  not 
in  reality  a  mixed  culture,  composed  of  a  resistant  bacterium  and 
a  bacteriophage.  One  must  be  sure  that  the  culture  is  not  only 
pure,  but  ultrapure. 

The  mutations  noted  by  different  authors  may  most  certainly 
be  attributed  to  the  frequency  of  these  mixed  cultures.  And 
the  confirmation  of  these  reports  has  been  lacking  for  the  very 
simple  reason  that  the  verification  has  been  attempted,  not  with 
strains  contaminated  by  the  bacteriophage,  but  with  cultures 
really  pure.  The  experimental  results  have  thus  very  naturally 
differed.  In  this  regard  I  might  say  that  it  is  indeed  singular, 
in  view  of  the  unique  character  of  certain  conceptions  concerning 
the  bacteriophage,  that  not  a  single  author  has  yet  traced  to  the 
fact  that  bacterial  cultures  are  frequently  contaminated  by  the 
bacteriophage,  the  conclusion  that  the  bacteriophage  takes  origin 
spontaneously  in  these  cultures.  In  reality,  as  we  see  it,  in  con- 
nection with  the  genesis  of  these  cultures,  each  time  that  there 
is  a  contamination  by  the  bacteriophage,  it  is  in  the  form  of  a 
mixed  culture.  The  bacteriophage  exists  in  the  culture  from 
the  beginning,  that  is,  from  the  time  of  isolation. 

In  the  experiments  touching  immunity,  the  contradictory 
results  may  likewise  well  reside  in  the  presence  of  a  bacteriophage 
in  the  cultures  employed  in  the  experiments.  We  will  see  in  the 


BACTERIOPHAGOUS  ANTISEBUM  143 

second  part  of  this  work,  the  role  which  the  bacteriophage  plays 
in  immunity,  and  it  is  evident  that  the  experimental  results  in 
immunological  investigation  will  be  entirely  different  if  the  bac- 
terial strain  employed  is,  or  is  not,  contaminated  by  the  bacterio- 
phage; whether  it  is  a  resistant  strain  or  a  normal  strain. 

In  a  word,  the  idea  of  the  existence  of  the  bacteriophage  im- 
poses the  obligation  of  always  verifying  the  bacterial  cultures 
with  a  view  to  determining  that  they  are,  not  simply  pure,  but 
ultrapure;  and  this  under  penalty  of  obtaining  entirely  false  ex- 
perimental results  as  a  result  of  the  possible  presence  of  the 
bacteriophage. 


CHAPTER  VI 

THE  NATURE  OF  THE  BACTERIOPHAGE 

Nature  of  the  Bacteriophage.  The  Number  of  Possible  Hypotheses. 
Experimental  Proofs  of  the  Living  Nature  of  the  Bacteriophage.  Refu- 
tation of  the  Hypothesis  of  Kabeshima.  Refutation  of  the  Hypothesis 
of  BordetandCiuca.  Refutation  of  the  Hypothesis  of  Bail.  Refutation 
of  the  Hypothesis  of  Salimbeni.  Conclusions. 

THE  NATURE  OF  THE  BACTERIOPHAGE 

All  of  the  facts  which  have  been  recognized  up  to  the  present 
time  and  which  have  been  recorded  in  the  preceding  chapters 
have  been  confirmed  by  all  authors  who  have  investigated  the 
question.  The  phenomena  themselves  have  never  been  the  sub- 
ject of  controversy,  and  because  of  their  definiteness,  it  might  be 
said  because  of  their  violence,  and  because  of  the  facility  with 
which  they  can  be  reproduced,  they  can  not  be  controverted. 

The  discovery  of  the  bacteriophage  was  associated  with  a  study 
of  a  disease  of  locusts,  in  which  I  for  the  first  time  noted  in  the 
intestine  of  the  insects  which  resisted  the  infection  a  principle 
antagonistic  to  the  action  of  the  pathogenic  cocco-bacillus;  a 
principle  which  could  be  demonstrated  by  its  effects  but  which 
of  itself  could  not  be  isolated.  With  this  suggestive  observation 
as  a  basis,  I  systematically  sought  for  a  comparable  principle  in 
the  intestinal  contents  of  patients  with  enteric  infections.  Finally, 
during  the  year  1915,  in  studying  an  epidemic  of  dysentery  which 
prevailed  in  a  squadron  of  cavalry  stationed  in  the  neighborhood 
of  Paris,  I  noted  the  phenomenon  of  plaques  in  the  cultures  on 
agar  tubes.  Shortly  after,  from  the  stools  of  a  patient  under 
treatment  in  the  Pasteur  Hospital  I  was  able  to  isolate  by  filtra- 
tion the  antagonistic  principle.  The  study  was  continued  dur- 
ing the  rare  moments  which  my  duties  as  Chief  of  the  Laboratory 
Service  for  the  Preparation  of  Vaccines  permitted.  (More  than 
twenty  million  doses  of  vaccines  were  furnished  by  the  Service 
to  the  Allied  Armies  during  the  war.)  I  tried  particularly  to 

144 


NATURE  OF  THE  BACTERIOPHAGE  145 

determine  the  nature  of  this  principle,  and  it  was  only  after  I 
had  considered  all  possible  a  priori  hypotheses,  multiplying  the 
control  experiments  which  had  demonstrated  experimentally 
with  certainty  that  the  principle  could  be  only  an  autonomous 
living  being, — a  filtrable  microbe  parasitizing  the  bacteria, — that 
I  resolved  to  publish,  in  1917,  the  first  communication  announc- 
ing the  discovery  of  an  ultramicrobe  parasitizing  the  dysentery 
bacillus.  In  this  report  I  gave  the  principal  characteristics  of 
the  virus  and  indicated  the  role  played  by  it  in  the  course  of  the 
disease.  From  1917  to  the  end  of  1919,  I  continued  these  in- 
vestigations alone,  extending  them  to  other  diseases,  and  it  was 
only  in  December  1919  that  Kabeshima,  working  in  my  labora- 
tory with  strains  which  I  had  furnished  him,  published  results 
which  confirmed  mine.  Since  that  time,  investigations  on  the 
bacteriophage  have  multiplied,  and  rare  indeed  are  the  labora- 
tories which  are  not  interested  in  the  question. 

It  may  seem  strange,  at  first  sight,  that  I  should  have  been 
able  to  work  alone  on  this  question  for  such  a  long  time;  a  cir- 
cumstance which  has  permitted  me  to  establish  the  facts  in  their 
entirety,  and  the  relation  between  them;  to  demonstrate  their 
importance  from  the  point  of  view  of  immunity,  and  to  accom- 
plish this  before  any  other  communication  appeared.  In  this  I 
have  been  favored  by  circumstances  and  even  by  the  strangeness 
of  the  facts  themselves,  which  at  first  excited,  not  merely  astonish- 
ment, but  incredulity,  even  among  my  most  friendly  colleagues, 
who  were  not  loath  to  consider  me  a  visionary.  This  time  has 
passed.  The  facts  are  recognized  to  be  correct.  The  most 
recent  work  appears  to  affirm  the  curative  properties  of  cultures 
of  the  bacteriophage.  The  only  point  at  issue  is  my  conception 
of  the  nature  of  the  active  principle. 

I  may  be  permitted  to  make  a  few  remarks  upon  the  subject  of 
this  discussion.  All  authors,  without  exception,  who  have  formu- 
lated an  hypothesis  regarding  the  nature  of  the  bacteriophage 
have  adopted  a  method  of  reasoning  that  is  somewhat  peculiar. 
None  of  them  have  taken  the  trouble  to  review  the  experiments 
that  I  had  accumulated  in  favor  of  the  living  nature  of  the  bac- 
teriophage during  the  years  that  I  had  alone  been  occupied  with 
this  question ;  experiments  moreover,  which  in  no  instance  are  open 


146  THE   BACTERIOPHAGE 

to  question.  Each  of  them  has  taken  simply  a  particular  fact, 
suited  to  support  his  thesis,  and  has  neglected  entirely  the  great 
group  of  experimental  facts  which  render  this  hypothesis  inad- 
missible, forgetting  that  that  which  accords  with  experiment  is, 
for  a  theory,  the  sole  and  indispensable  criterion  of  its  truth. 

But  the  strangeness  of  their  procedure  is  not  restricted  to  the 
interpretation  of  their  experimental  findings,  but  extends  to  the 
experiments  themselves.  These  experiments  correctly  performed 
react  against  their  hypotheses.  I  have  called  attention  to  these 
errors  and  Kabeshima  seems  to  be  converted  by  the  evidence, 
for  he  has  published  nothing  for  two  years.  As  for  Bordet,  he 
does  not  maintain  that  his  fundamental  experiment,  called  that 
of  leucocytic  exudates,  may  be  repeated.  He  has  recognized 
that  the  specificity  of  the  bacteriophage,  a  condition  sine  qua 
non  for  his  hypothesis,  is  contrary  to  fact,  but  he  nevertheless 
continues  to  support  a  hypothesis  thenceforth  without  foundation. 

THE   POSSIBLE  HYPOTHESES 

The  number  of  possible  hypotheses  is  limited,  and  after  a  con- 
sideration of  these  fundamental  hypotheses  it  is  only  necessary 
to  select  that  which  accords  with  the  observed  experimental  facts 
which  have  been  contradicted  by  no  one.  These  hypotheses 
were  carefully  reviewed  prior  to  all  publication  in  an  attempt  to 
determine  the  nature  of  the  principle  which  I  had  discovered. 

Three  fundamental  hypotheses  can  be  formulated  and  any 
other  view  must  be  a  modification  or  a  combination  of  one  or 
more  of  these  three.  Discussion  of  these  three  must  necessarily 
dispose  of  any  subsidiary  hypotheses  that  may  be  advanced. 

What,  then,  are  these  three  fundamental  hypotheses  which 
comprise  all  that  can  possibly  be  formulated? 

First  hypothesis 

The  bacteriophage  is  derived  from  the  superior  organism  in 
its  reaction  to  the  bacterial  invasion  by  the  production  of  a  prin- 
ciple which  provokes  the  destruction  of  the  bacterium. 

This  first  hypothesis  admits  of  two  solutions. 

1.  The  principle  in  question  is  a  substance  of  diastatic  nature. 
The  single  fact  of  the  serial  action  of  the  principle  is  sufficient  to 


NATURE   OF   THE    BACTERIOPHAGE  147 

reject  this  explanation,  for  such  a  substance  would  be  rapidly 
eliminated  in  the  successive  dilutions  during  repeated  transfers. 
It  is  thus  useless  to  discuss  this  further.  Up  to  the  present  time 
no  one  has  recommended  this. 

2.  The  active  principle  derived  from  the  organism  reacting 
against  the  infection  is  particulate,  an  organic  being,  capable  of 
developing  afterward  outside  of  the  organism  at  the  expense  of 
the  bacteria. 

This  hypothesis  does  not  constitute  a  scientific  heresy,  for  it 
is  not  contradicted  by  any  experimental  fact.  In  the  case  of 
any  hypothesis,  however  improbable  it  may  appear  in  view  of  the 
actual  state  of  biologic  science,  if  it  can  not  be  experimentally 
demonstrated  false  and  if  it  harmonizes  with  the  demonstrated 
facts,  it  can  not  be  rejected  a  priori.  Moreover,  Carrel  has  shown 
that  it  is  possible  to  cultivate  tissues  outside  of  the  organism; 
and  in  addition,  Altmann  has  proposed  a  theory  according  to 
which  the  zymogenic  granulations  can  be  nothing  but  bioplasts, 
independent  elements,  having  their  individual  existence  and 
capable  of  reproduction  by  division  in  a  cellular  medium.  The 
bacteriophage  may  be  a  bioplast,  derived  from  the  superior  organ- 
ism, and  capable  of  multiplication  at  the  expense  of  the  bacteria. 

However  this  may  be,  since  this  particle,  this  "organite," 
comports  itself  from  the  time  when  it  is  taken  from  the  organism 
as  an  autonomous  being  capable  of  assimilation  and  reproduction, 
and  since  it  acts  as  a  being  corresponding  to  the  definition  of  a 
microbe,  it  must  be  a  minute  being  endowed  with  life.  We  will 
revert  to  this  idea  in  the  case  of  the  third  hypothesis. 

Second  hypothesis 

The  bacteriophage  may  be  derived  from  the  lysed  bacterium 
itself. 

The  two  subsidiary  hypotheses  given  above  may  again  be 
formulated:  1.  The  bacteria  secrete  a  diastase  with  autolytic 
functions.  As  we  will  see,  this  is  in  effect  the  conception  of 
Kabeshima.  Bordet  takes  over  this  hypothesis  with  an  added 
complication,  since  he  explains  the  origin  of  the  principle  in  terms 
of  the  first  hypothesis,  that  is,  a  substance  derived  from  the  or- 
ganism, and  explains  the  continuity  in  series  by  means  of  the 


148  THE   BACTERIOPHAGE 

second  hypothesis,  that  is,  to  a  substance  derived  from  the  bac- 
terium itself. 

This  hypothesis  fails  before  the  following  experimental  facts: 

a.  The  bacteriophage  exists  in  the  form  of  particles  which 
multiply. 

b.  The  bacteriophage  does  not  exercise  a  specific  action  upon 
any  single  species  of  bacteria,  but  at  one  and  the  same  time,  the 
same  bacteriophage  may  be  active  against  several  species. 

c.  All  strains  of  the  bacteriophage,  whether  they  be  active 
against  the  staphylococcus,  against  the  dysentery  bacillus,  against 
B.  pestis,  or  against  any  other  organism,  belong  to  the  same  species, 
as  demonstrated  by  the  complement  fixation  reaction. 

2.  The  bacteriophage  is  derived  from  the  bacterium,  but  is 
particulate,  an  "organite"  capable  of  life,  conducting  itself  thence- 
forth like  an  autonomous  ultramicrobe.  This  is  the  hypothesis 
of  Bail. 

While  an  interpretation  which  comprehends  an  "  organite" 
derived  from  the  superior  parasitized  organism  does  not  neces- 
sarily imply  specificity  of  the  bacteriophage,  any  hypothesis  in- 
volving an  "  organite"  derived  from  the  bacterium  which  is  sub- 
jected to  lysis  does  imply  a  strict  specificity.  The  last  view, 
therefore,  is  inadmissible,  since  experiment  proves  that  a  single 
strain  of  the  bacteriophage  may  be  active  toward  several  species 
of  bacteria  at  once,  and  that  all  strains  of  the  bacteriophage  be- 
long to  the  same  species. 

All  possible  hypotheses,  save  that  of  the  ultramicrobe,  which 
we  will  shortly  examine,  can  only  be  some  combination  of  the 
two  preceding  ones.  All,  then,  will  be  subject  to  the  same  ob- 
jections; none  will  be  admissible  for  it  will  be  contradicted  by 
experimental  facts. 

Third  hypothesis 

The  bacteriophage  is  an  autonomous  organism,  an  ultramicrobe 
parasitizing  the  bacteria.  This  hypothesis  is  the  only  one  which 
accords  with  all  the  recorded  experimental  facts,  and  it  is  for 
this  logical  reason  that  I  have  attached  myself  to  it.  For  it,  I 
have  acquired  more  and  more  convincing  evidence  which  I  have 
not  been  able  to  disprove,  for  the  numerous  new  facts  that  I 


NATURE   OF  THE   BACTERIOPHAGE  149 

have  discovered  harmonize  with  this  hypothesis,  with  this  hy- 
pothesis only,  and  none  contradict  it. 

Moreover,  I  should  repeat  that  I  have  not  formed  any  hypothesis 
as  to  the  species  to  which  the  ultramicrobe  belongs.  I  have 
called  it  Bacteriophagum  intestinale,  a  name  simply  denoting  its 
characteristic  property  and  the  place  where  I  first  found  it.  Is 
it  a  protozoan?  Is  it  a  bacterium?  Does  it  belong  to  a  kingdom 
which  is  neither  vegetable  nor  animal?  Does  it  arise  even  in 
another  organism,  a  possibility  which  has  been  suggested  in  ex- 
amining the  first  hypothesis?  These  questions  can  be  ignored. 
It  is  an  ultramicrobe,  a  filtrable  being  endowed  with  the 
functions  of  assimilation  and  of  reproduction,  functions  which 
characterize  the  living  nature  of  beings  and  which  pertain  to 
them  alone.  That  is  all  that  experimentation  is  actually  able 
to  demonstrate. 

To  try  to  penetrate  further  into  its  identity  would  be  nothing 
but  purely  speculative  reasoning. 

EXPERIMENTAL  PROOFS  OF  THE  LIVING  NATURE  OF  THE 
BACTERIOPHAGE 

This  section  will,  without  doubt,  be  judged  unnecessary  by 
the  reader  who  has  followed  the  experiments  recorded  in  the 
preceding  chapters.  However,  it  may  be  well  to  group  these 
proofs  and  to  comment  on  certain  experiments  which  can  leave 
no  doubt,  even  in  the  minds  of  the  most  skeptical  and  uninformed. 

1.  The  bacteriophage  proliferates,  since  serial  cultures  can  be 
continued  indefinitely.     In  the  action  of  the  diastases  there  is 
always  a  certain  proportionality.     The  action  is  the  more  ener- 
getic when  the  amount  employed  is  large.     With  the  bacterio- 
phage this  is  not  true.     The  activity  is  due  to  the  quality  of  the 
principle,  not  to  its  quantity,  and  this  is,  indeed,  a  property  of 
vital  activity.     A  diastase  acts  in  proportion  to  its  quantity,  a 
bacterium  in  proportion  to  its  virulence. 

2.  The  bacteriophage  presents  properties  analogous  to  those 
of  other  known  living  beings.     Its  resistance  to  agents  of  de- 
struction, although  great,  is,  however,  less  than  that  of  many 
organisms   of   which   the   living   nature  is  unquestionable   and 
uncontroverted. 


150  THE   BACTEEIOPHAGE 

I  ought  in  this  connection  to  dwell  upon  the  action  of  tempera- 
ture, since  some  authors  have  suggested  that  the  temperature 
of  destruction  of  the  bacteriophage  was  too  high  to  allow  a  con- 
sideration of  them  as  living  beings.  It  is  thus  necessary  to  re- 
call some  of  the  elementary  facts  which  readers  of  this  work  cer- 
tainly ought  not  to  ignore.  The  lethal  temperature,  as  we  have 
seen,  is  about  75°C.  Without  mentioning  the  living  organisms 
from  thermic  sources,  of  which  the  temperature  reaches  up  to 
93°C.,  it  is  known  that  one  may  readily  isolate  from  sewage  bac- 
teria which  develop  normally  at  a  temperature  of  75°C.  More- 
over, Duclaux  has  shown  that  the  young  cells  of  Tyrothrix  tennis 
do  not  die  until  a  temperature  of  about  100°C.  has  been  reached. 
A  lethal  temperature  of  75°C.,  far  from  being  exceptional,  must 
be  recognized  as  well  below  that  resisted  by  a  large  number  of 
unicellular  organisms. 

In  so  far  as  the  action  of  antiseptics  is  concerned,  the  bacterio- 
phage takes  a  position  intermediate  between  the  bacteria  in  their 
vegetative  form  and  the  spores  derived  from  these  bacteria. 
More  resistant  than  the  first,  they  are  more  sensitive  than  the  sec- 
ond. Compared  from  this  point  of  view  with  other  known  ultra- 
microbes,  they  are  definitely  more  susceptible  than  some.  The 
virus  of  the  tobacco  mosaic,  for  example,  will  resist  for  several 
months  a  concentration  of  alcohol  which  will  kill  the  bacteriophage 
in  a  short  time.  The  virus  of  rabies,  and  that  of  vaccinia, 
remain  alive  in  concentrations  of  glycerine  that  destroy  the 
bacteriophage. 

It  is  to  be  noted  that  the  bacteriophage  presents  the  character- 
istic of  being  particularly  sensitive  to  certain  reagents  which 
have  absolutely  no  effect  upon  the  diastases;  quinine  for  example. 
As  for  glycerine,  which  destroys  the  bacteriophage,  it  constitutes 
the  medium  of  choice  for  the  indefinite  preservation  of  bacterial 
toxins  and  the  most  sensitive  diastases. 

3.  With  sufficiently  active  strains  of  the  bacteriophage  a  com- 
plete and  permanent  lysis  is  secured;  all  of  the  bacteria  contained 
in  a  suspension  are  definitely  destroyed.  Moreover,  serial 
passages  of  the  bacteriophage  are  possible  in  bacterial  suspensions 
made  in  fluids  which  do  not  permit  the  development  of  these 
bacteria; — physiological  salt  solution,  or  bouillon  with  forty  per 


NATURE   OF  THE   BACTERIOPHAGE  151 

cent  glycerine,  for  example.  This  proves  again  that  the  survival 
of  a  certain  number  of  bacteria  does  not  constitute  a  factor  in 
the  serial  activity,  for,  as  this  factor  would  fail  the  series  could 
not  be  continued. 

4.  On  agar  the  bacteriophage  gives  colonies  at  the  expense  of 
the  bacteria,  and  this  permits  counting  the  active  elements.  A 
soluble  ferment,  diastase  or  toxin,  can  not  concentrate  its  action 
in  definite  points.  It  may  be  objected  that  the  lytic  diastase 
is  provided  by  the  bacteria  themselves  and  that  each  plaque  on 
agar  represents  the  area  where  is  to  be  found,  after  the  inocula- 
tion, a  bacterium  particularly  able  to  furnish  the  diastase  under 
the  influence  of  a  force  "X." 

The  following  experiments  demonstrate  that  this  objection  is 
not  valid. 

Experiment  LV.  (A}.  Take  10  tubes.  Into  the  first  place  10  cc.  of  a 
suspension  of  B.  dysenteriae  Shiga  containing  100,000,000  bacilli  per  cc., 
into  the  second  place  10  cc.  of  a  suspension  containing  200,000,000  bacilli 
per  cc.,  into  the  third  place  10  cc.  of  a  300,000,000  suspension,  and  so  on, 
increasing  by  100,000,000  the  concentration  of  the  suspensions  introduced 
into  each  tube  of  the  series.  The  tenth  tube,  then,  will  have  a  suspension 
containing  1,000,000,000  bacilli  per  cc.  Each  of  these  tubes  is  then  inocu- 
lated with  an  equal  quantity  of  a  very  dilute  culture  of  the  bacteriophage 
filtered  through  a  bougie,  about  0.000005  cc.  We  have  then  a  series  of  10 
tubes  containing  a  more  and  more  concentrated  suspension  of  B.  dysenteriae 
Shiga  in  the  ratio  of  1 : 2: 3: 4: 5: 6: 7:8: 9: 10,  and  an  equal  amount  of  bacterio- 
phage culture.  The  tubes  are  carefully  shaken,  and  0.02  cc.  from  each  tube 
is  planted  upon  agar  slants.  After  incubation  at  37°C.,  each  of  the  10 
tubes  of  agar  shows  a  culture  of  B.  dysenteriae  spotted  with  plaques,  and 
the  number  of  these  plaques  is  practically  the  same  for  all  the  tubes. 

(B)  Take  10  tubes,  each  containing  10  cc.  of  a  suspension  containing 
100,000,000  B.  dysenteriae  per  cc.,  that  is,  a  like  suspension  in  all  10  tubes. 
To  the  first  add  1/100,000  cc.  of  filtered  bacteriophage  culture,  to  the 
second  1/200,000  cc.,  to  the  third  1/300,000  cc.,  1/400,000  cc.  to  the  fourth, 
and  so  on,  each  tube  receiving  a  smaller  and  smaller  amount  of  bacterio- 
phage culture,  so  that  the  tenth  tube  will  contain  only  1/1,000,000  cc. 
Thus,  we  have  a  series  of  10  tubes,  all  containing  an  equal  number  of  dysen- 
tery bacilli  and  an  amount  of  bacteriophage  culture  varying  according  to 
the  ratio  10:9:8:7:6:5:4:3:2:1.  Shake  the  tubes  thoroughly  and  plant 
0. 02  cc.  from  each  of  the  10  suspensions  on  to  agar  slants.  After  incubation 
at  37°C.  each  of  the  agar  tubes  will  show  a  covering  growth  of  B.  dysenteriae 
studded  with  plaques,  but  the  number  of  plaques  in  the  tubes  varies  with 
the  proportion  of  bacteriophage  culture  which  has  been  added.  Prac- 


152  THE   BACTEKIOPHAGE 

tically,  their  number  varies  from  tube  1,  which  had  received  the  largest 
amount  of  bacteriophage  culture,  to  tube  10,  which  had  received  the 
smallest,  in  the  proportion  of  10:9:8:7:6:5:4:3:2:1. 

These  two  experiments,  which  complement  each  other,  demon- 
strate unquestionably,  that  the  active  principle  is  contained 
solely  in  the  filtered  culture  of  the  bacteriophage;  and  that  this 
active  principle  is  composed  of  material  elements,  capable  of 
forming  colonies  on  agar  at  the  expense  of  the  surrounding  bac- 
teria in  such  a  way  that  their  enumeration  is  possible.  These 
material  elements  are  capable  of  multiplication  as  is  shown  by 
the  formation  of  colonies  and  by  action  in  series.  It  can  thus 
only  be  a  living  organism.  This  experiment  by  itself  is  sufficient 
to  demonstrate  that  the  bacteriophage  is  a  "  formed  ferment," 
which  in  reality  implies  the  existence  of  an  ultramicrobe  parasitic 
of  the  bacteria. 

5.  Eliava  and  Pozerski  have  shown  that  toward  concentra- 
tions of  free  H  and  OH  ions  the  range  fatal  for  the  bacteriophage 
is  more  limited  than  for  the  bacteria.     The  diastases  and  toxins 
react  in  a  wholly  different  fashion. 

6.  Dumas,  confirmed  by  Beckerich  and  Hauduroy,   have  iso- 
lated the  bacteriophage  from  the  soil  and  from  the  filtered  water 
of  streams.     I  have  myself  isolated  it  from  sea-water.    There  is 
nothing  strange  in  this,  since  it  is  an  ultramicrobe  derived  from 
stools.     This  fact  can  not,  on  the  contrary,  be  reconciled  with  a 
hypothesis  of  a  ferment,  whether  the  ferment  be  of  leucocytic  or 
other  origin. 

7.  Diastases  in  solution  are  absorbed  by  the  precipitates  which 
form  upon  the  addition  of  alcohol.    This  takes  place  with  cul- 
tures of  the  bacteriophage,  but  the  elements  which  precipitate  are 
not  the  ultramicrobes  themselves.    The  ultramicrobes  are  de- 
stroyed by  the  alcohol,  and  the  principle  which  is  precipitated 
will  not  reproduce  the  action  in  series.     This  possibility  of  ex- 
tracting from  a  culture  an  active  principle  which  can  only  be  a 
secretory  product  of  the  bacteriophage  shows  indeed  that  the 
latter  can  be  nothing  other  than  a  living  being. 

8.  I  have  shown  that  the  bacteriophage  is  capable  of  adaptation 
to  the  harmful  action  of  glycerine.     Adaptation  is  the  appanage 
of  living  beings  exclusively. 


NATURE   OF  THE   BACTERIOPHAGE  153 

9.  It  is  impossible  to  isolate  two  strains  of  the  bacteriophage 
which  are  identical  in  the  intensity  of  their  action  or  in  the  scope 
of  their  activity.     With  a  single  strain  the  intensity  of  the  ac- 
tion can  be  varied  experimentally.     Variation  is  an  exclusive 
characteristic  of  life. 

10.  The  lytic  action  is  always  exercised  by  one  and  the  same 
element  as  is  demonstrated  in  the  complement  fixation  reaction. 
This  element  adapts  itself  to  parasitism  toward  such  and  such  a 
bacterium,  and  the  possibility  of  such  adaptation  necessarily 
implies  the  living  nature  of  the  element  which  exercises  it. 

11.  Bruynoghe  and  Maisin  have  shown  that  the  bacteriophage 
is  phagocytized   and   destroyed  by  the  leucocytes.     This  fact 
shows  that  the  bacteriophage  is  foreign  to  the  organism,  and  it 
alone  demonstrates  that  it  can  not  be  of  leucocytic  origin. 

The  nature  of  the  bacteriophage  is  not  open  to  question;  its 
origin  alone  may  be  disputed.  Is  it  an  entirely  autonomous  being, 
a  "species",  botanically  or  zoologically?  Is  it  a  "bioplast" 
capable  of  indefinite  reproduction  at  the  expense  of  living  bac- 
teria, conducting  itself  as  an  autonomous  being  of  which  it  has 
all  the  properties? 

It  is  still  impossible  to  decide  this;  experiment  alone  will  de- 
termine. However  this  may  be,  the  two  hypotheses,  although 
they  may  differ  as  to  the  origin  of  the  bacteriophage,  agree  as  to 
its  nature.  The  most  probable  interpretation  is  that  the  ultra- 
microbe  is  autonomous,  a  botanic  or  zoologic  "species." 

REFUTATION  OF  THE  HYPOTHESIS  OF  KABESHIMA 

Without  doubt  readers  will  wish  to  know  the  diverse  hypotheses 
proposed  by  those  who  have  opposed  the  living  nature  of  the 
bacteriophage,  and  I  will  discuss  them  in  their  chronological 
order,  although  this  may  not  be  their  documentary  standing. 

Apart  from  the  confirmation  of  the  experiments  which  I  alone, 
or  with  my  collaborators,  have  effected,  these  discussions  repre- 
sent about  all  that  has  been  done  on  the  question  of  the  bacterio- 
phage. By  indicating  these  and  commenting  on  them,  this  work 
becomes  completed  and  is  a  comprehensive  statement  of  the 
present  knowledge  regarding  the  bacteriophage. 


154  THE   BACTERIOPHAGE 

Kabeshima,  in  1920,  starting  on  the  one  hand  from  considera- 
tions without  significance  (as,  for  example,  the  thermal  death 
point  of  the  bacteriophage,  which  he  found  to  be  too  high  to  be 
applied  to  living  beings),  and  on  the  other  hand,  from  inexact 
experimental  results  (he  announced,  for  example,  that  the  bac- 
teriophage resisted  the  action  of  alcohol;  that  it  was  active  in  the 
presence  of  sodium  fluoride,  etc.),  formulated  the  following 
hypothesis.  Under  the  action  of  a  proferment  playing  the  role 
of  a  catalyzer,  the  autolytic  diastases  are  activated  and  bring 
about  their  dissolving  action. 

We  have  already  considered  the  principal  objections  which 
render  this  hypothesis  untenable.  It  fails  to  explain  serial  ac- 
tion, for  a  proferment  would  disappear  rapidly  as  a  result  of  dilu- 
tion in  the  course  of  passages;  it  does  not  take  account  of  the  fact 
that  the  bacteriophage  presents  itself  in  the  form  of  autonomous 
particles  capable  of  being  counted;  it  is  formally  contradicted 
by  the  fact  that  the  same  bacteriophage  can  act  on  diverse  bac- 
terial species,  etc.  The  inadmissibility  of  the  hypothesis  of 
Kabeshima  has  been  recognized,  moreover,  by  all  authors  who 
have  considered  the  question. 

KEFUTATION  OF  THE  HYPOTHESIS  OF  BORDET  AND  CIUCA 

Bordet  and  Ciuca  (October,  1920)  very  significantly  modified 
the  hypothesis  of  Kabeshima  to  the  end  of  explaining  serial 
activity.  They  said: 

"In  view  of  the  fact  that  the  stools  of  patients  with  dysentery  are  rich  in 
leucocytes,  and  that  the  lysinogenic  power  is  only  observed  toward  the 
period  of  convalescence,  we  have  asked  ourselves  if  the  phenomenon  of 
d'Herelle  is  not  the  result  of  a  defensive  activity  of  the  organism,  and 
particularly  of  an  activity  of  the  leucocytic  exudate.  This  produces  in 
the  bacterium  an  hereditary  nutritive  vitiation,  consisting  in  the  produc- 
tion by  the  bacterium  of  a  sort  of  lytic  ferment,  which  is  capable,  moreover, 
of  diffusing  in  the  ambient  fluid  and  as  a  result,  reacting  in  the  same  fashion 
on  normal  bacteria  of  the  same  species." 

This  proposition  takes  no  account  of  the  previously  established 
facts.  Among  other  facts  it  disregards  that  I  had  made  it  known 
some  time  previously  that  the  bacteriophage  was  a  normal  in- 
habitant of  the  intestine,  and  that  lysogenic  power  was  also  to 


NATURE   OP  THE   BACTERIOPHAGE  155 

be  observed  at  times  other  than  at  the  moment  of  convalescence. 
At  a  time  considerably  earlier,  I  had  likewise  indicated  that  a 
strain  of  the  bacteriophage  exercised  its  action  not  only  against 
a  single  bacterial  species,  but  against  several  at  the  same  time. 
Like  the  hypothesis  of  Kabeshima,  this  of  Bordet  and  Ciuca  takes 
no  account  of  the  fundamental  fact,  already  demonstrated  ex- 
perimentally, of  the  existence  of  the  bacteriophage  in  the  form 
of  particles  which  it  is  possible  to  count. 

Fundamentally,  how  does  this  hypothesis  of  Bordet  and  Ciuca 
differ  from  that  of  Kabeshima?  It  differs  in  nothing  except  it 
be  in  the  form  in  which  it  is  stated.  It  hinges  upon  an  arbitrary 
transposition  of  effect  and  cause;  a  transposition  upon  which 
they  lay  no  stress.  The  leucocytic  exudate  induces  the  "  nutri- 
tive vitiation"  (?)  which  results  in  the  lysis  of  the  bacteria  in 
the  first  tube  of  the  series,  but  in  the  following  ones  the  same  effect 
will  be  produced,  no  longer  by  the  leucocytic  exudate,  which 
will  of  necessity  have  disappeared  in  the  first  tubes  because  of 
the  dilution,  but  by  the  bacterial  lytic  ferment  alone.  Bordet 
and  Ciuca  seem  to  find  this  substitution  of  cause  entirely  logical, 
when  in  reality  it  is  contrary  to  all  that  we  know.  In  admitting 
a  priori,  that  a  liquid,  indeed  a  filtered  liquid,  is  able  to  transport 
with  it  an  hereditary  property,  there  is,  it  appears,  an  affirmation 
which  must  needs  be  based  on  experiment  and  not  solely  upon 
the  inference  of  Bordet  and  Ciuca.  And  what  could  have  been 
the  fundamental  experiments  which  suggested  such  conclusions? 
They  say: 

"If  one  or  two  days  after  the  last  injection,  one  removes  by  puncture  the 
peritoneal  exudate,  rich  in  leucocytes,  from  a  guinea  pig  which  had  received 
three  or  four  intraperitoneal  injections  of  B.  coli  at  intervals  of  a  few  days, 
one  can  demonstrate  that  this  exudate,  when  added  to  normal  bacteria  of 
the  same  species,  modifies  them,  and  confers  upon  them  a  very  pronounced 
autolytic  power,  transmissible  from  culture  to  culture." 

They  add  that  they  will  shortly  publish  the  results  secured  with 
other  bacterial  species.  These  results,  promised  more  than  a 
year  and  a  half  ago,  have  not  yet  been  furnished.  Furthermore, 
not  having  succeeded  in  reproducing  the  experiment  with  the 
leucocytic  exudate,  in  spite  of  numerous  attempts,  I  refuted  the 
statements  of  Bordet  and  Ciuca,  in  a  note  published  in  the  Compt. 


156  THE   BACTERIOPHAGE 

rend.  Soc.  de  biol.  This  contradiction  has  remained  without  a 
reply  for  more  than  eight  months — evidence  that  these  authors 
themselves  have  not  succeeded  in  repeating  the  experiment. 

Furthermore,  the  experiment  with  the  leucocytic  exudate, 
had  it  been  correct,  would  in  no  case  have  provided  proof  for  the 
non-reality  of  the  bacteriophagous  ultramicrobe,  for  it  would  not 
have  disproved  any  of  the  experiments  which  demonstrate  its 
reality,  and  of  more  significance,  it  accords  perfectly  with  the 
idea  of  a  parasitic  ultramicrobe.  Long  before  Bordet  and  Ciuca 
worked  with  the  bacteriophage  I  had  demonstrated  that  there 
was  in  certain  cases  a  passage  of  the  intestinal  bacteriophage  into 
the  circulation,  and  that  it  could  be  isolated  from  the  blood. 
Since  the  bacteriophage  may  acquire  by  adaptation  the  faculty 
of  parasitizing  any  bacterial  species,  and  since  the  bacteriophage 
is  a  normal  inhabitant  of  the  body  of  all  animals,  it  is  by  no  means 
impossible  that  one  might  experimentally  succeed  in  provoking 
in  the  body  of  an  animal  this  adaptation  for  a  given  bacterium 
which  has  passed  into  the  circulation.  We  will  see  in  Part  II 
of  this  work  that  this  is  exactly  the  series  of  phenomena  which 
occur  in  natural  disease;  and  I  can  not  see  in  what  respect  the 
fact  of  the  experimental  reproduction  of  this  sequence  of  events 
will  be  opposed  to  the  doctrine  of  an  ultramicrobe  parasitizing 
the  bacteria. 

I  may  say  further,  that  even  had  their  experiments  been  cor- 
rect, the  logical  interpretation  would  not  have  been  that  of  Bordet 
and  Ciuca;  that  the  bacteriophagous  principle  is  derived  from 
the  leucocytes.  In  fact,  if  the  primum  movens  of  the  bacterioly- 
sis transmissible  in  series  resides  in  the  leucocytes,  it  should  be 
enough,  for  example,  to  add  to  a  suspension  of  B.  dysenteriae 
the  leucocytes  from  a  horse  furnishing  an  anti-dysentery  serum, 
that  is,  from  an  hyperimmunized  horse,  to  reproduce  the  phenom- 
enon of  lysis  transmissible  in  series.  This  reaction  would  have 
been  recognized  long  ago  by  innumerable  investigators,  perhaps 
first  of  all  by  Bordet,  who  for  more  than  thirty  years  has  in- 
vestigated the  antibodies  in  the  blood  of  immunized  animals. 
This  experiment  I  have  in  vain  attempted  many  times,  well 
before  Bordet  and  Ciuca  announced  their  hypothesis,  when  I  was 
attempting  to  test  all  possible  hypotheses  touching  the  origin 


NATURE   OF  THE   BACTEEIOPHAGE  157 

of  the  principle  dissolving  the  bacteria.  Far  from  possessing 
the  ability  to  start  serial  lysis,  the  leucocytes  derived  from  a 
horse  hyperimmunized  with  the  dysentery  bacillus,  do  not  even 
intervene  to  inhibit  the  growth  of  this  bacillus,  whatever  may  be 
the  quantity  of  leucocytes  employed  in  the  test. 

Finally,  had  the  experiment  of  the  leucocytic  exudate  been 
correct  its  interpretation  would  not  in  any  case  have  served  as  a 
proof  for  the  reality  of  an  ''hereditary  nutritive  vitiation"  trans- 
mitting itself  by  means  of  a  liquid  factor.  To  be  tenable,  an 
hypothesis  must  take  into  account  all  the  facts,  and  that  of  Bordet, 
exactly  like  that  of  Kabeshima  from  which  it  is  copied,  is  incom- 
patible with  the  experimental  facts  which  I  have  reported.  It 
implies,  among  other  things,  the  strict  specificity  of  the  bacterio- 
phage.  Even  if  one  admits  as  possible  the  entirely  speculative 
hypothesis  of  "  hereditary  nutritive  vitiation"  transmitted  by 
the  intervention  of  a  liquid,  one  is  absolutely  unable  to  admit  that 
this  liquid  is  able  to  transmit  the  "  hereditary  vitiation' '  from  one 
bacterial  species  to  another  bacterial  species.  Moreover,  Bor- 
det and  Ciuca  at  first  maintained  that  there  was  such  a  strict 
specificity.  However,  in  view  of  the  accumulated  evidence  they 
have  recognized  that  the  action  of  the  bacteriophage  is  not 
specific,  but,  in  spite  of  this,  they  have  not  abandoned  their 
conception  or  even  offered  anything  by  way  of  explanation. 

The  accidental  positive  result  obtained  by  Bordet  and  Ciuca 
in  the  experiment  with  the  leucocytic  exudate  can  readily  be  ex- 
plained in  perfect  harmony  with  the  doctrine  of  the  ultramicrobial 
bacteriophage.  Such  an  explanation  is,  that  the  intestinal  bac- 
teriophage has  passed  into  the  peritoneal  cavity  of  the  experi- 
mental guinea  pig  as  a  result  of  the  irritation  produced  there  by 
the  injections.  This  has  been  followed  by  a  growth  of  the  bac- 
teriophage in  this  cavity  at  the  expense  of  the  bacteria  injected. 
As  we  will  see  in  Part  II,  the  bacteriophage  does  not  remain  con- 
fined to  the  intestinal  tract;  it  is  able  to  enter  the  circulation. 
Moreover,  the  experiment  of  Bordet  regularly  becomes  positive, 
even  if  the  experimental  guinea  pig  has  received  only  one  pre- 
liminary injection  of  bacteria,  provided  one  or  two  cubic  centi- 
meters of  a  culture  of  the  bacteriophage  active  for  the  bacterium 
inoculated  is  administered  per  os  a  few  hours  before  the  intra- 


158  THE   BACTERIOPHAGE 

peritoneal  injection.  This  experiment  is  adequate  to  give  a 
correct  interpretation  to  the  accidental  finding  obtained  by  Bor- 
det  and  Ciuca. 

To  summarize:  the  hypothesis  of  Bordet  and  Ciuca  dealing 
with  the  nature  of  the  bacteriophage  is  inadmissible,  first,  because 
it  does  not  conform  to  the  experimental  facts;  second,  because 
it  is  founded  upon  an  erroneous  interpretation  of  an  experiment 
which  has  not  been  repeated,  and  which,  even  if  it  had  been  cor- 
rect, would  not  have  served  as  a  basis  upon  which  such  an  hy- 
pothesis could  be  founded;  third,  because  invoking  as  an  explana- 
tion an  hereditary  phenomenon,  it  is  in  formal  contradiction  to 
all  known  facts  concerning  the  hereditary  transmission  of 
characters. 

Bruynoghe  and  his  collaborators,  who  at  the  beginning  of  their 
studies  adopted  the  point  of  view  of  Bordet,  have  since  realized 
that  the  experimental  facts  do  not  harmonize  with  this  hypothesis. 
They  now  support  the  idea  of  the  ultramicrobe,  a  parasite  of  the 
bacteria. 

REFUTATION  OF  THE  HYPOTHESIS  OF  BAIL 

Bail,  in  1921,  rejected  the  hypothesis  of  Kabeshima  and  of 
Bordet.  According  to  him  the  bacteriophage  existed  indeed  in 
the  form  of  autonomous  masses  as  I  had  demonstrated.  It  con- 
ducted itself  as  an  ultramicrobe  but  these  particles  could  only  be 
constituted  by  the  "splitter,"  that  is  to  say,  by  particles  derived 
from  the  lysed  bacteria  themselves.  These  organized  particles, 
capable  of  reproduction  under  a  filtrable  form  at  the  expense  of 
the  same  bacteria,  secreted  a  dissolving  diastase. 

Bail  adduces  in  favor  of  his  hypothesis  the  fact  that  he  has 
been  able  to  isolate  from  old  cultures  a  bacteriophage  active  for 
the  dysentery  bacillus  of  Flexner.  The  cause  of  Bail's  error  is 
easy  to  detect.  He  has  dealt  with  mixed  cultures.  I  have 
already  called  attention  to  the  frequency  of  such  cultures  and 
I  have  indicated  their  origin. 

The  hypothesis  of  Bail  corresponds  exactly  to  the  second  solu- 
tion of  the  second  possible  hypothesis  discussed  above.  It  is 
needless  to  repeat  the  refutation  which  has  been  presented.  Let 
us  simply  recall  that  the  condition  sine  qua  non  for  the  validity 


NATURE   OF  THE   BACTERIOPHAGE  159 

of  this  hypothesis  would  be  strict  specificity,  and  experiment 
demonstrates  that  this  does  not  obtain.  All  authors  are  actu- 
ally in  accord  on  this  point  and  have  confirmed  the  observation 
that  a  single  bacteriophage  is  able  to  attack  diverse  bacterial 
species. 

REFUTATION  OF  THE  HYPOTHESIS  OF  SALIMBENI 

The  hypothesis  of  Salimbeni  is  merely  mentioned.  Admitting 
that  the  bacteriophage  is  an  autonomous  microorganism,  he 
considered  it  a  Myxomycete,  presenting  itself  in  visible  forms, 
and  visible  even  to  the  naked  eye.  His  observations  have  been 
contradicted  by  all  who  have  studied  the  bacteriophage.  More- 
over, Salimbeni  himself,  has  not  continued  to  maintain  this  hy- 
pothesis. Possibly  the  observation  upon  which  he  based  his 
hypothesis  was  due  to  the  use  of  contaminated  cultures. 

CONCLUSIONS 

All  of  the  authors  who  have  advanced  hypotheses  other  than 
that  of  an  ultramicrobe  parasitizing  the  bacteria  have  forgotten 
that  a  hypothesis  ought  always  to  account  for  the  entire  mass  of 
facts;  that  it  is  necessary,  not  only  to  demonstrate  that  it  suffices 
to  justify  the  phenomena,  but  it  must  also  prove  that  these  phe- 
nomena can  not  be  justified  if  this  hypothesis  is  abandoned  or  if 
it  is  modified. 

After  all,  the  whole  controversy  on  the  subject  of  the  nature 
of  the  bacteriophage  is  only  the  renewal  of  the  old  discussion 
which  we  had  for  a  long  time  thought  terminated.  With  the  new 
ideas  of  diastases  capable  of  multiplication,  or  of  co-ferments  or 
catalyzers  playing  with  the  metaphysics  of  ubiquity,  or  of  se- 
cretory immunity  transmissible  by  communicated  motion;  it 
is  only  taking  up  anew  the  old  theory  of  Stahl,  that  "any  body 
brought  to  a  state  of  putrefaction  transmits  very  easily  this 
state  to  another  body  still  free  of  corruption."  This  theory  of 
the  multiplication  of  a  principle  of  communicating  motion  had 
been  held  by  Liebig  in  his  discussion  with  Pasteur  concerning 
the  mechanism  of  fermentation.  Pasteur  demonstrated  experi- 
mentally that  it  was  false,  and  we  were  lead  to  believe  the  fact 
definitely  acquired.  With  vital  phenomena  or  communicating 


160  THE   BACTERIOPHAGE 

motion,  the  discussion  and  the  experiments  of  demonstration  hinge 
on  the  same  facts;  we  only  descend  a  step  in  the  order  of  magni- 
tude of  the  beings  concerned. 

This  same  discussion  may  perhaps  be  renewed  again  some  day 
if  we  descend  still  another  step;  if  we  discover,  for  example,  a 
virus  parasitizing  the  bacteriophage.  The  infinitely  small  is  as 
conceivable  as  the  infinitely  great;  we  have  not  the  right  to  assign 
limits  to  them. 


PART  II 

THE  ROLE  OF  THE  BACTERIOPHAGE 
IN  IMMUNITY 


INTRODUCTION 

Up  to  the  present  time  investigations  on  immunity  have  been 
directed  toward  solving  the  following  question:  What  are  the 
means  of  defense  which  permit  an  immunized  animal  or  one 
naturally  refractory  to  resist  infection?  These  studies  have 
resulted  in  the  development  of  diverse  theories. 

When  an  animal  is  affected  with  a  contagious  disease  of  bac- 
terial origin,  the  cellular  immunity,  which  we  call  "organic 
immunity,"  abstracting  it  from  all  theory  as  to  its  intimate  na- 
ture, is  only  established  in  a  variable  length  of  time  after  the 
inception  of  the  disease.  Does  the  animal  remain  without  de- 
fense up  to  the  time  that  this  organic  immunity  becomes  effec- 
tive? By  what  phenomenon  is  it  possible  to  acquire  this  organic 
immunity? 

All  animals  sensitive  to  an  infection  and  exposed  to  it  do  not 
contract  the  disease.  Why  do  some  of  them  remain  unharmed? 

These  are  the  principal  points  upon  which  the  experiments  to 
be  discussed  have  turned.  As  will  be  seen,  they  lead  to  a  new 
chapter  in  the  study  of  the  means  of  defense  against  infection; 
and  the  conclusions  themselves  which  will  be  derived  from  these 
investigations  will  not  actually  contradict  anything  in  the  present 
theories,  for  they  apply  to  different  states. 

There  is,  nevertheless,  a  particular  point  in  the  present  theories 
of  immunity  to  which  I  wish  to  call  attention,  namely,  that  which 
deals  with  bacteriolysis  as  induced  by  specific  sera.  This  is 
certainly  pertinent  since  it  deals  with  the  subject  under  dis- 
cussion:— the  lysis  of  bacteria. 

Everyone  knows  the  nature  of  the  phenomenon  of  Pfeiffer.  If 
one  injects  a  suspension  of  cholera  vibrios  into  the  peritoneal 
cavity  of  a  guinea  pig  previously  "immunized,"  a  transformation 
of  these  vibrios  into  granules  is  noted.  Pfeiffer  has  suggested 
that  the  transformation  into  granules  constitutes  only  the  first 
phase  of  the  vibriolysis;  but  in  this  he  was  in  error,  for  the  granules 
maintain  this  form  indefinitely. 

163 


164  INTRODUCTION 

We  have  very  frequently  sought  for  the  act  of  disappearance  of  the 
granules  in  drops  taken  from  the  peritoneal  fluid,  but  the  number  of  these 
transformed  vibrios  has  never  diminished,  even  after  several  days,  and  we 
have  therefore  not  been  able  to  detect  the  phenomenon  of  dissolution  of 
the  granules.  In  spite  of  all  this,  it  is  incontestible  that  the  granular 
transformation  is  a  manifestation  of  a  very  grave  change  to  which  the 
cholera  vibrios  have  been  subjected  under  the  influence  of  the  peritoneal 
fluid  of  the  immunized  organism.1 

Is  this  granular  transformation,  as  Metchnikoff  states,  a  mani- 
festation of  very  grave  lesions?  The  fact,  established  by  him, 
that  one  of  these  granules  seeded  on  agar  gives  a  colony  of  nor- 
mal vibrios  is  not  an  index  of  a  very  profound  alteration.  Let 
us  consider  for  the  moment  that  there  can  not  be,  in  any  sense, 
a  lysis  of  the  cholera  vibrios  in  the  Pfeiffer  reaction.  One  may 
try  by  all  sorts  of  methods  to  provoke  the  same  phenomenon  in 
all  kinds  of  animals  with  all  kinds  of  bacteria,  but  without  result. 
It  can  only  be  secured  with  the  cholera  vibrios;  they  alone  can  be 
transformed  into  granules. 

Metchnikoff,  and  later  Bordet,  showed  that  the  reaction  of 
Pfeiffer  could  likewise  take  place  in  vitro.  To  obtain  a  granular 
transformation  it  is  only  necessary  to  introduce  the  vibrios  into 
the  fresh  serum  of  an  immunized  animal.  Bordet  further 
showed  that  this  transformation  was  brought  about  by  the  in- 
teraction of  two  principles,  amboceptor  and  alexin. 

The  amboceptor,  thermostabile,  is  specific;  that  is  to  say,  it  is 
only  active  toward  the  element  against  which  the  animal  fur- 
nishing the  serum  has  been  immunized.  It  exists  only  in  traces, 
or  not  at  all,  in  the  serum  of  normal  animals.  It  develops  as  an 
effect  of  immunization. 

The  alexin,  thermolabile,  is,  it  appears,  common.  It  exists  in 
as  large  an  amount  in  a  normal  animal  as  in  the  immunized 
animal.  It  is  fixed  by  any  element  previously  acted  upon  by  a 
specific  amboceptor. 

Bordet  next  discovered  that  the  blood  of  an  animal  prepared 
by  the  injection  of  the  red  blood  cells  of  a  different  animal  species 
formed  specific  amboceptor.  If  to  a  suspension  of  these  cells 
is  added  a  heated  serum  of  the  treated  animal  (thus  containing 

1  Metchnikoff.  Uimmuniii  dans  les  maladies  infectieuses,  Paris,  1901, 
Masson  et  Cie. 


INTRODUCTION  165 

the  amboceptor)  and  then  some  fresh  serum  from  a  normal  animal 
(thus  containing  alexin)  the  phenomenon  of  hemolysis  is  obtained, 
— a  phenomenon  in  which  the  dissolution  of  the  red  cells  is  simu- 
lated, although  in  reality,  as  Bordet  himself  showed,  there  is 
simply  a  diffusion  of  the  hemoglobin,  the  stroma  remains  intact. 

Finally,  Bordet  showed,  in  an  indirect  manner,  by  means  of 
the  complement  fixation  reaction  which  bears  his  name,  that 
bacteria,  as  well  as  red  blood  cells,  absorb  specific  amboceptor 
and  are  thus  able  to  fix  complement. 

Here  is  where  the  equivocation  commences.  By  analogy  it 
was  concluded  that  since,  under  the  influence  of  the  complement 
fixed  by  the  cells  in  combination  with  the  specific  amboceptor, 
a  hemolysis  of  the  red  cells  was  produced,  so  with  bacteria  which 
likewise  absorb  a  specific  amboceptor  and  fix  complement  in  the 
same  way,  there  must  necessarily  be  a  bacteriolysis.  This  bac- 
teriolysis has  never  been  observed  directly,  and  this  fact  is  ade- 
quate, it  seems  to  me,  to  arouse  some  doubt  concerning  the  reality 
of  the  phenomenon.  The  following  experiment,  which  I  have 
repeated  several  times,  shows  that  in  reality  the  bacteria  are  by 
no  means  destroyed  under  such  conditions.  The  result  is  quite 
the  opposite. 

Experiment  LVI.  Take  4  tubes,  each  containing  20  cc.  of  0.8  per  cent 
saline.  To  each  add  a  quantity  of  cholera  vibrio  suspension  sufficient  to 
give  a  count  of  about  1000  vibrios  per  cubic  centimeter. 

The  first  tube  remains  as  the  control. 

The  second  tube  receives  0.25  cc.  of  fresh  guinea  pig  serum,  after  this 
serum  has  been  shown  by  test  to  contain  complement. 

The  third  tube  receives  0.1  cc.  of  an  anticholera  serum,  in  which  the 
presence  of  specific  amboceptor  has  been  demonstrated. 

The  fourth  tube  receives  0.25  cc.  of  the  fresh  guinea  pig  serum  and  0.1 
cc.  of  the  anticholera  serum.  Thus,  in  this  tube,  the  vibrios  are  in  the 
presence  of  a  specific  antibody  and  of  complement.  The  4  tubes  are  incu- 
bated at  37°C.,  and  from  day  to  day  are  tested  to  see  if  the  vibrios  are  alive 
or  dead.  To  this  end,  after  twenty-four  hours,  the  tubes  are  thoroughly 
shaken  and  from  each  of  them  2.5  cc.  is  immediately  taken  and  planted 
into  a  tube  of  sterile  bouillon.  The  first  of  the  tubes  usually  (4  times  in  6) 
remains  sterile2  while  the  other  three  give  cultures  of  the  cholera  vibrio, 

2  This  bactericidal  action  of  physiological  saline  is  rather  strange.  Even 
when  working  with  relatively  concentrated  suspensions,  containing  from 
50  to  100  million  bacteria  (cholera  vibrios  or  B.  dysenteriae)  per  cubic 


166  INTRODUCTION 

normal  in  the  second  tube  containing  complement  only,  agglutinated  in 
the  last  two. 

After  forty-eight  hours  transfers  are  again  made.  The  first  tube  always 
remains  sterile,  the  second  is  often  sterile  (3  times  in  6),  the  last  two  give 
agglutinated  cultures. 

After  three  days  the  subcultures  result  as  follows ;  the  first  two  tubes  are 
always  sterile,  the  third  often  so  (4  times  in  6),  the  last  always  gives  an 
agglutinated  culture. 

After  four  days  the  first  three  tubes  are  always  sterile.  The  last  tube 
only,  that  is,  the  one  containing  both  antibody  and  complement,  gives  an 
agglutinated  culture. 

After  six  days  the  same  result  is  obtained. 

The  same  experiment  has  been  performed  with  the  Shiga  dys- 
entery strain  and  the  anti-dysentery  serum  of  the  Pasteur  In- 
stitute. The  result  was  in  all  respects  comparable.  The  Shiga 
bacillus,  like  the  cholera  vibrio,  persists  for  a  long  time  in  the 
suspension  containing  the  anti-serum  and  the  alexin. 

Variations  in  all  directions  have  been  made  in  the  proportions 
of  the  sera,  both  in  that  containing  the  complement,  and  in  that 
containing  the  antibody,  as  well  as  in  the  concentration  of  the 
bacterial  suspension.  The  results  have  all  been  essentially  the 

centimeter,  the  sterilization  is  complete  in  a  few  hours  at  a  temperature  of 
37°C.  On  the  contrary,  these  organisms  will  remain  alive  for  some  days 
in  tap  water.  And  what  is  still  more  singular,  is  that  everyone  has  adopted 
physiological  saline  for  the  preparation  of  bacterial  suspensions,  concluding 
a  priori,  that  bacteria  must  be  preserved  alive  for  a  long  time  in  a  medium 
spoken  of  as  "isotonic." 

At  the  bottom  of  this  we  find  a  false  deduction  by  comparison.  Eed 
blood  cells  hemolyze  in  a  few  seconds  in  tap  water,  but,  on  the  contrary, 
they  resist  hemolysis  in  isotonic  saline  solution.  Thus,  it  is  concluded, 
without  doubt,  that  bacterial  cells  must  conduct  themselves  in  the  same 
manner.  As  a  matter  of  fact,  this  is  absolutely  contrary  to  what  takes 
place. 

As  we  will  see,  the  same  reasoning  has  been  held  with  regard  to  the  so- 
called  bacteriolysis  with  sera.  There  also,  one  falls  into  an  error,  and  this 
will  always  be  the  case  when  an  attempt  is  made  to  substitute  deduction 
based  on  analogy  for  experimentation,  especially  when  the  elements  con- 
cerned are  as  different  as  a  bacterium  and  a  red  cell. 

With  reference  to  the  toxic  action  of  sodium  chloride,  Loeb  (Biochem. 
Zeitschr.,  1906,  2,  81)  has  shown  that  this  salt  may  be  toxic  for  all  the 
unicellular  organisms  living  in  the  sea,  and  that  this  toxicity  may  be  neu- 
tralized by  the  salts  of  potassium  and  calcium. 


INTRODUCTION  167 

same.  The  bacteria  always  remain  alive  in  the  antibody-com- 
plement mixture  for  a  much  longer  time  than  in  pure  physiological 
saline. 

These  experiments  show  that  cholera  vibrios,  or  dysentery 
bacilli,  are  rapidly  destroyed  in  saline;  that  they  remain  alive 
longer  in  the  presence  of  a  normal  fresh  serum  containing  com- 
plement or  in  an  antiserum;  and  that  they  remain  alive  still 
longer  in  the  presence  of  both  the  antiserum  and  complement. 
It  is  therefore  evident  that  the  bacteria,  sensitized  and  having 
fixed  complement,  far  from  being  subjected  to  lysis,  are  more 
resistant  than  normal  bacilli. 

The  sera  termed  "  antibacterial"  do  not,  in  vitro  at  least,  play 
any  bacteriolytic  role.  Everything  indicates  that  it  is  the  same 
in  vivo.  We  know  that  at  times  the  antisera,  derived  from  horses 
thoroughly  immunized  by  the  injection  of  living  bacilli,  are  con- 
taminated by  bacilli  of  the  same  species  as  those  which  have  been 
injected,  and  that  it  is  possible  to  demonstrate  them  by  culture. 
Anti-rouget  serum  provides  a  remarkable  example.  The  serum 
from  a  horse  hyperimmunized  by  a  series  of  injections  of  living 
culture  possesses  extremely  marked  curative  and  preventive 
properties,  but  it  is,  nevertheless,  rather  frequently  contaminated 
by  the  bacillus  of  rouget,  even  if  the  serum  is  withdrawn  some  ten 
to  twelve  days  after  an  injection.  How  can  we  reconcile  this 
fact,  which  indeed  is  not  an  isolated  observation,  with  the  hypothe- 
sis that  the  immunity  which  it  confers  is,  by  some  mechanism 
at  present  unexplained,  associated  with  the  presence  of  an  " anti- 
bacterial antibody?"  If  it  should  produce  bacteriolysis,  it  cer- 
tainly ought  to  do  so  in  the  hyperimmunized  animals  themselves, 
where  the  bacterium  finds  itself  in  contact  with  an  abundance  of 
antibody  and  of  complement. 

On  the  other  hand,  sera  very  rich  in  antibody  may  entirely 
lack  preventive  power,  and  the  opposite  is  also  true.  Metchni- 
koff  and  his  collaborators  have  furnished  many  examples  of  this. 

If  we  pass  to  a  consideration  of  natural  immunity  we  readily 
discern  that  there  is  absolutely  no  parallelism  between  the  state 
of  the  patient  and  the  antibody  content  of  the  blood.  In  human 
typhoid  fever,  for  example,  the  fatal  relapses  may  occur  when  the 
antibody  is  at  its  maximum.  Finally,  in  diseases  which  result 


168  INTRODUCTION 

in  immunity  the  antibody  disappears  a  short  time  after  the  at- 
tack, but  this  does  not  prevent  the  persistence  of  immunity  for 
years,  or  even  decades,  after  the  disappearance  of  the  antibody. 

To  summarize :  however  the  serum  of  hyperimmunized  animals 
or  the  serum  of  an  individual  affected  with  some  infectious  disease 
may  act,  it  is  impossible  to  establish  any  relation  between  the 
antibacterial  antibodies — so-called — and  immunity.  These  anti- 
bodies, like  the  agglutinins,  can  only  be  considered  as  indices 
of  infection. 

It  may  be  objected  that  preventive  vaccination  by  a  sensitized 
virus  indicates  a  fragility  in  the  bacteria  impregnated  with  anti- 
body. This  objection  is  in  reality  not  an  objection,  for  experi- 
mental facts  show,  on  the  contrary,  that  a  sensitized  bacterium 
is  as  virulent  as  a  normal  bacterium.  I  have  proved  this  for 
B.  typhi  murium  with  the  mouse,  and  for  the  bacterium  of  bovine 
hemorrhagic  septicemia  in  cattle.  These  animals  are  killed  in 
the  same  time  and  by  the  same  dose,  whether  the  bacteria  in- 
jected are  normal  or  sensitized.  The  possibility  of  injecting  man, 
without  great  inconvenience,  with  sensitized  cholera  vibrios  or 
sensitized  typhoid  bacilli  (and  it  may  still  be  said  with  some  re- 
serve in  this  last  case)  does  not  negative  these  findings  at  all, 
seeing  that  Ferran  has  given  tens  of  thousands  of  preventive 
vaccinations  against  cholera,  using  living  cultures,  and  that  Ch. 
Nicolle  has  demonstrated  the  possibility  of  vaccinating  man 
against  typhoid  fever  by  injecting  him  with  living,  normal 
typhoid  bacilli.  In  general,  injections  of  sensitized  bacteria  are 
no  more  inoffensive  than  injections  of  the  normal  living  organ- 
isms, and  they  are  equivalent,  since  living  sensitized  bacteria 
and  living  normal  bacilli  are  virulent  to  the  same  degree. 

One  cannot  avoid  the  conclusion  that  it  is  impossible  to 
attribute  any  active  role  in  the  production  of  antibacterial  im- 
munity to  any  actually  known  antibodies.  All  organic  immunity 
is  reduced  to  antibacterial  immunity,  assured  by  phagocytosis, 
and  to  antitoxic  immunity,  assured  by  the  antitoxins.3 

But  is  this  organic  immunity  an  attribute  of  the  immunized 
animal  only?  Is  it  not  enjoyed  by  a  susceptible  animal?  All 

3  Meaning  by  antitoxins  all  antibodies  which  neutralize  a  soluble  toxic 
substance. 


INTRODUCTION  169 

individuals  exposed  to  an  infection  do  not  contract  the  disease, 
and  to  what  do  they  owe  this  privilege?  Once  diseased,  the 
immunity  in  the  susceptible  animal  manifests  itself  only  after  a 
lapse  of  time,  which  in  the  most  favorable  cases  is  hardly  less 
than  twelve  days.4  Does  the  animal  remain  without  defense 
during  this  lapse  of  time?  Finally,  in  this  animal  affected  by 
disease,  whatever  may  be  the  mechanism  of  organic  immunity, 
how  can  this  immunity  be  established  to  render  this  sick  animal 
refractory?  Under  what  influence  is  phagocytosis  released? 
Under  what  influence  do  the  antitoxins  originate? 

The  role  which  the  partisans  of  the  theory  of  "bactericidal 
humoral  immunity"  have  desired  that  these  simple  indices  of 
infection — the  antibodies — play,  is  based  upon  a  non-existent 
phenomenon  of  bacteriolysis  by  immune  sera.  Could  it  not  in 
reality  be  played  by  the  bacteriophage,  that  principle  endowed 
with  a  powerful  bacteriolytic  action,  operating  upon  the  most 
varied  bacteria? 

Could  not  the  bacteriophage  play  a  role  in  the  defense  of  the 
organism,  a  preponderant  role  in  the  susceptible  animal,  and  as 
such,  deprived  of  all  acquired  immunity?  In  other  words,  does 
there  not  exist  by  the  side  of  the  homogeneous  organic  immunity, 
an  immunity  originating  in  the  bacteriophagous  ultramicrobe, 
and,  as  a  result,  an  heterogeneous  immunity? 

We  have  seen  in  a  preceding  chapter  that  the  lysin  of  the  bac- 
teriophage may  possess  an  extraordinarily  potent  opsonic  activity. 
Can  not  the  bacteriophage  play,  in  addition  to  its  direct  action, 
an  important  role  in  phagocytosis  itself,  in  bringing  about  what 
might  be  called  a  phagocytic  education? 

4  Animals  vaccinated  by  an  attenuated  anthrax  or  rouget  virus  are  not 
protected  against  natural  infection  until  after  this  period  of  time.  I  have 
also  shown  that  at  least  twelve  to  fifteen  days  are  necessary  to  secure  an 
immunity  following  the  use  of  an  attenuated  virus  in  bovine  homorrhagic 
septicemia.  In  typhoid  fever,  the  immunity  acquired  as  a  result  of  infec- 
tion is  even  longer  in  establishing  itself,  as  the  possibility  of  relapse  in 
well-advanced  convalescence  shows.  This  lapse  of  time,  twelve  days, 
is  therefore  a  minimum  insofar  as  naturally  acquired  organic  immunity 
is  concerned.  There  is  nothing  to  be  gained  here  by  discussing  laboratory 
experiments  concerning  the  development  of  immunity  in  refractory  ani- 
mals, for  such  experiments  are  laboratory  phenomena  only  and  have  nothing 
necessarily  in  common  with  natural  conditions. 


170  INTRODUCTION 

Finally,  in  dissolving  the  bacteria,  can  it  not  be  an  indirect 
factor  in  naturally  acquired  antitoxic  immunity? 

These  are  the  points  which  we  will  consider  in  the  second  part 
of  this  monograph. 

It  would  seem  that  the  only  method  which  ought  to  be  followed 
in  investigating  the  relation  between  immunity  and  a  principle 
to  which  one  may  attribute  a  protective  power  ought  to  be  founded 
on  the  observation  of  natural  disease,  and  that  the  parallelism 
between  the  state  of  the  patient  and  the  presence,  and  the  potency, 
of  the  supposed  protective  principle,  ought  to  serve  as  the  cri- 
terion for  determining  its  true  role.  If  a  parallelism  exists,  it 
may  be  regarded  in  the  possible  relation  of  cause  and  effect,  and 
one  can  then  turn  to  the  counter-test  for  confirmation.  If  a 
parallelism  does  not  exist,  the  relation  of  cause  and  effect  cannot 
be  invoked,  and  the  principle  under  consideration  cannot  play 
an  active  role  in  the  processes  of  recovery.  This  is  the  method 
of  investigation,  the  only  logical  one  it  seems  to  me,  that  I  have 
applied  in  investigating  the  relationship  between  the  bacterio- 
phage  and  immunity.  I  consider,  in  fact,  that  a  theory  of  im- 
munity based  only  on  simple  observation  or  on  comparison,  always 
remains  subject  to  discussion.  For  simple  observation  readily 
leads  to  error,  especially  when  the  observations  are  made  on 
refractory  animals  and  are  not  found  to  be  confirmed  when  applied 
to  a  susceptible  animal.  It  is  certainly  much  easier  to  experi- 
ment in  the  laboratory  with  caged  animals;  to  study  the  immunity 
against  the  cholera  vibrio,  for  example,  on  a  guinea  pig  which  is 
resistant  to  the  disease  naturally,  than  to  run  everywhere  in 
search  of  epizootics  in  order  to  study  the  disease  in  its  normal 
environment.  But  common  sense  alone  is  adequate  to  make  it 
apparent  that  the  first  method  can  prove  nothing,  and  that  only 
observation  of  the  natural  disease,  complemented  by  experimen- 
tation on  an  animal  susceptible  to  it,  can  give  results  that  have 
an  absolute  value.  It  may  indeed  seem  strange  that  we  use  the 
word  "  immunization"  in  speaking  of  a  refractory  animal,  since 
the  refractory  state  already  represents  immunity  carried  to  its 
highest  degree. 

I  wish  to  be  free  of  such  criticism  and  will  thus  follow  an  order 
which  seems  to  me  the  most  logical.  We  will  observe  first, 


INTRODUCTION  171 

natural  infection  and  we  will  see  if  the  search  for  the  bacterio- 
phage  and  the  determination  of  its  properties  at  different  stages 
of  the  disease  and  of  convalescence  provides  results  which  have 
any  relation  to  the  pathologic  condition  of  the  patient.  I  have 
selected  for  this  investigation  different  infections,  enteric  and 
septicemic,  diseases  of  man  and  of  animals,  with  which  we  will 
show  that  defense  by  the  bacteriophage  is  a  phenomenon  of  a 
general  nature.  Some  of  the  diseases  studied  are  epidemic,  and 
we  will  have  occasion  to  note  the  effect  of  the  bacteriophage  on 
the  progress  of  the  epidemic  itself. 

If  the  bacteriophage  is  an  agent  of  immunity,  it  will  not  appear 
only  at  the  exact  moment  when  it  is  most  needed.  It  should  be 
a  normal  inhabitant  of  the  intestine.  We  will  look  for  it,  then, 
in  the  healthy  individual,  choosing  subjects  throughout  all  animal 
species,  and  this  will  show  the  generality  of  the  presence  of  the 
bacteriophage. 

Finally,  we  will  attempt  the  counter-test.  If,  in  the  susceptible 
animal  the  principle  of  antibacterial  immunity  resides  in  the 
bacteriophage,  the  administration  to  a  susceptible  animal  of  a 
bacteriophage  active  for  a  given  bacterium  ought  to  render  the 
organism  resistant  to  the  disease  caused  by  this  bacterium. 

Thanks  to  the  kindness  of  M.  Roux,  Director  of  the  Pasteur  In- 
stitute, and  to  M.  Yersin,  Director  of  the  Pasteur  Institute  in 
Indo-China,  I  have  been  able  to  accomplish  in  its  entirety  the 
program  which  I  have  outlined. 

In  France  I  have  had  the  opportunity  to  study  the  role  of  the 
bacteriophage  in  intestinal  diseases,  and  during  the  course  of  a 
year  spent  in  the  Pasteur  Institute  at  Saigon,  I  have  been  able 
to  verify  the  generality  of  the  phenomena  observed,  by  a  study 
of  a  highly  contagious  septidemia, — barbone  in  the  buffalo, — 
and  by  a  disease  of  glandular  localization, — plague. 

It  is  certain  that  a  theory  of  immunity  based  on  the  bacterio- 
phage, that  is,  on  an  autonomous  organism,  is  so  far  outside  of 
all  present  opinion  that  it  will  stir  up  at  first  incredulity  and  will 
be  called  a  "finalistic  theory," — a  synonym  of  "  anti-scientific." 
I  affirm  that  from  my  point  of  view  this  theory  can  not  be  "final- 
istic."  "To  be  is  to  struggle,  to  live  is  to  conquer,"  a  very  just 
statement  by  Le  Dantec.  It  is  all  contained  in  a  single  word — 


172  INTRODUCTION 

evolution.  A  being  which  evolves  is  necessarily  a  being  which 
lives,  which  adapts  itself,  and  which  conquers.  From  the  instant 
that  it  ceases  to  adapt  itself — to  evolve — it  dies.  Evolution  is 
always  conducted  according  to  the  law  of  least  effort.  The  multi- 
cellular  organisms  have  profited  by  securing  for  their  defense 
the  parasitism  of  the  bacteriophage  for  the  bacteria;  which  is 
only  a  chapter  in  the  universal  struggle. 

If,  among  all  living  beings,  the  bacteria  alone  escaped  para- 
sitism, where  would  we  arrive?  It  is  very  simple.  One  of  two 
things  would  take  place.  Either  evolution  would  not  extend 
beyond  the  stage  of  the  unicellular  being,  or  evolution  would  be 
accomplished  in  another  manner  and  immunity  would  be  as- 
sured by  other  means;  a  simple  matter  of  adaptation.  The  bac- 
teriophage does  not  exist  for  the  defense  of  the  superior  organ- 
ism against  the  bacteria,  it  exists  simply  because  in  the  course  of 
evolution  certain  germs  have  parasitized  others. 

Nothing  in  nature  exists  simply  for  an  end,  for  nature  is  not 
an  end.  That  there  exist  on  the  earth  thinking  beings,  or  that 
they  might  not  have  been,  is  a  perfectly  negligible  incident.  Is 
this  point  of  view  "finalistic"?  But  what  does  it  matter;  a  scien- 
tific theory  is  true  or  false  according  to  the  proofs  upon  which  it 
is  founded. 

Each  time  that  we  will  speak  in  the  course  of  this  discussion 
of  "antibacterial  immunity"  it  is  essential  to  understand  "anti- 
bacterial immunity  in  a  susceptible  individual."  These  observa- 
tions and  experiments,  as  I  have  already  remarked,  are  concerned 
with  this  and  this  alone. 

Up  to  the  present  I  have  paid  little  attention  to  the  phenomena 
of  immunity  in  the  refractory  animal.  It  indeed  seems,  in 
general,  in  the  special  type  of  immunity  which  characterizes  the 
refractory  state,  that  the  elimination  of  bacteria  which  may  gain 
access  to  the  body,  and  which  because  of  the  refractory  state  are 
not  pathogenic,  is  effected  by  phagocytosis.  In  this  special 
case,  defense  by  the  bacteriophage  could  not  possess,  the  greater 
part  of  the  time,  the  opportunity  to  act.  Phagocytosis  is  ac- 
complished too  rapidly  to  allow  the  bacteriophage  time  to  in- 
crease its  virulence  toward  the  bacterium  which  is  introduced 
into  the  organism. 


CHAPTER   I 

THE  BACTERIOPHAGE  IN  DISEASE 

Choice  of  Diseases  to  Study.  Bacillary  Dysentery.  B.  coli  Infections. 
Typhoid  and  Paratyphoid  Fevers.  Avian  Typhosis.  Barbone.  Bu- 
bonic Plague.  Flacherie.  Conclusions. 

CHOICE   OF  DISEASES   TO   STUDY 

From  the  point  of  view  of  the  study  of  immunity  human  infec- 
tion offers  an  inconvenience.  Man  is  not  available  for  experi- 
mentation; observation  alone  is  permitted.  On  the  other  hand, 
the  study  of  a  human  infection,  such  as  typhoid  fever  or  cholera 
for  example,  in  a  refractory  animal — and  they  are  all  so — can 
only  lead  to  illusory  results.  Study  of  disease  in  the  animal,  on 
the  contrary,  permits  of  confirmatory  experimentation  upon 
the  susceptible  animal  itself  where  error  is  no  longer  unavoidable. 
However,  this  method  of  procedure  is  very  complicated;  the  dis- 
ease does  not  come  to  us,  we  must  go  to  it. 

The  study  of  typhoid  fever  and  of  dysentery  allows  us  to  show 
by  observation  the  role  of  the  bacteriophage  in  the  course  of  the 
disease.  These  same  phenomena  may  be  reproduced  in  the 
course  of  infection  in  animals,  and  it  is  possible  with  the  latter 
to  conduct  such  experiments  of  verification  as  will  confirm  that 
which  simple  observation  has  already  shown. 

In  order  to  ascertain  the  influence  of  the  bacteriophage  on  the 
morbid  state,  a  method  which  consists  in  investigating  at  random 
the  activity  of  the  bacteriophage  in  a  specimen  of  material  taken 
at  any  time  whatsoever  will  not  lead  to  any  result.  It  is  neces- 
sary to  take  the  patient  as  quickly  as  possible  after  the  inception 
of  the  disease  and  to  examine  the  feces  each  day  until  recovery 
is  complete.  The  daily  findings  are  then  plotted  in  a  curve  which 
is  superimposed  on  that  expressing  the  general  state  of  the  indi- 
vidual, such  as  the  number  of  stools  in  dysentery,  or  the  tem- 
perature in  typhoid.  A  comparison  of  these  two  curves  allows 
one  to  draw  a  conclusion.  This  mode  of  procedure  necessitates 

173 


174  THE   BACTEBIOPHAGE 

considerable  work,  but  it  must  be  applied  for  it  is  the  only  proce- 
dure which  will  allow  of  a  conclusion. 

We  have  seen  in  the  course  of  the  preceding  chapters  that  the 
virulence  of  a  strain  of  the  bacteriophage  is  rarely  limited  to  any 
one  particular  bacterial  species,  but  exercises  in  general,  with 
variable  intensity,  its  action  on  several  species  pertaining  to  the 
same  group  or  to  closely  related  groups. 

It  would  be  practically  impossible,  in  view  of  the  length  of  the 
operations,  to  investigate  all  of  the  bacteria  which  may  be  attacked 
by  a  bacteriophage  isolated  from  the  stools  of  a  patient  at  any 
given  time.  The  procedure  must,  therefore,  be  reduced  to  a 
systematic  examination  in  each  case  of  the  virulence  of  the 
bacteriophage  toward  the  particular  bacterium  involved  in  a 
causal  relationship.  The  virulence  should  be  determined  for  a 
type  strain  of  this  bacterium  which  has  been  cultivated  for  a 
long  time  in  the  laboratory,  for  the  strain  derived  from  the  pa- 
tient himself,  and  for  B.  coli.  Eventually,  the  investigation 
may  be  extended  to  bacteria  belonging  to  the  same  group  or  to 
related  groups. 

We  know  that  the  virulence  of  different  strains  of  bacteriophage 
for  a  given  bacterium  is  far  from  constant.  It  varies  throughout 
a  scale  which  goes  from  zero  to  an  activity  such  that  it  is  suffi- 
cient to  add  only  a  few  germs  to  a  heavy  suspension  of  this 
bacterium  in  order  to  obtain  within  three  or  four  hours  a  complete 
and  permanent  lysis,  all  the  bacteria  being  then  destroyed.  Be- 
tween these  two  limits, — no  virulence  and  extreme  virulence — 
all  intermediate  degrees  are  possible.  A  weak  virulence  we  have 
seen,  may  be  enhanced  in  vitro,  but  in  so  far  as  the  study  of  im- 
munity is  concerned,  the  point  in  which  we  are  interested  is  the 
virulence  presented  by  the  bacteriophage  in  the  organism  at  the 
moment  of  observation;  or  the  actual  virulence  of  the  bacterio- 
phage contained  in  the  filtrate  prepared  directly  from  the  feces 
at  any  given  time  during  the  disease. 

As  we  have  also  seen,  the  appearance  of  cultures  of  the  bacterio- 
phage in  bouillon  or  on  agar  enables  us  to  evaluate  its  virulence 
for  the  bacterium  in  question.  In  order  to  facilitate  explanation 
in  the  further  discussion  of  the  subject  we  will  adopt  a  scale  of 
virulence  fixed  as  follows: 


THE   BACTERIOPHAGE   IN   DISEASE  175 

0  =  no  virulence  toward  a  given  bacterium.  Normal 
cultures  of  the  bacterium  develop  in  bouillon 
or  on  agar,  whatever  the  quantity  of  the  fil- 
trate from  the  feces  which  had  been  added. 
+  =  weak  virulence.  The  growth  in  bouillon  of 
the  bacterium  to  which  the  filtrate  has  been 
added  is  apparently  normal.  Transfer  of  this 
culture  to  agar  gives,  after  incubation,  a  culture 
layer  showing  a  few  minute  plaques.  Some 
of  the  bacteriophagous  germs  have  therefore 
attacked  the  bacteria  and  have  formed  colonies. 
+  +  =  medium  virulence.  The  culture  of  the  bacterium 
to  which  the  filtrate  has  been  added  is  almost 
normal  in  bouillon.  Transfers  of  this  cul- 
ture to  agar  give,  after  incubation,  either  a 
culture  layer  of  the  bacterium  studded  by  very 
numerous  colonies  of  the  bacteriophage,  pre- 
senting an  appreciable  surface  area,  or  of 
fragments  of  bacterial  culture  because  of  the 
very  great  number  of  bacteriophage  colonies, 
high  virulence.  Lysis  of  a  bacterial  suspension 
is  obtained  but  secondary  cultures  constantly 
develop.  The  reinoculations  on  to  agar  remain 
sterile  or  give  only  rare  colonies  of  the  bacterium, 
extreme  virulence.  The  bouillon  suspension 
shows  complete,  and,  in  general,  permanent 
lysis.  Inoculations  on  to  agar  always  remain 
sterile. 

Obviously,  it  would  be  possible  to  establish  a  more  detailed 
scale  of  virulence.  (Moreover,  this  has  been  done  in  the  curves 
which  will  be  given,  where  the  interval  between  no  virulence  and 
extreme  virulence  has  been  subdivided  into  ten  steps,  in  accord- 
ance with  the  aspect  of  the  cultures,  the  number  of  colonies  of 
the  bacteriophage,  and  the  size  of  the  plaques,  which  bear  a  rela- 
tion to  its  virulence.)  Practically,  the  appreciation  is  adequate 
with  four  steps,  particularly  in  view  of  the  fact  of  the  extreme 
variability  of  virulence  in  the  bacteriophage  in  the  body  of  a 
single  individual  from  one  time  to  another. 


176  THE  BACTERIOPHAGE 

The  expression  "Shiga  +  +  ++,  Hiss  +,  Flexner  0,  Typhoid 
++,  Para  A  0,  Para  B  0,  B.  coli  +  ++"  means,  then,  that  the 
bacteriophage  contained  in  the  filtrate  derived  from  the  stool 
of  an  individual  presents  an  extreme  virulence  for  B.  dysenteriae 
Shiga,  a  weak  virulence  for  B.  dysenteriae  Hiss,  an  average  viru- 
lence for  B.  typhosus,  and  a  high  virulence  for  B.  coli,  with  none 
for  B.  dysenteriae  Flexner  or  for  the  paratyphoids  A  or  B. 

BACILLARY  DYSENTERY 

The  subjoined  curves  show,  much  better  than  any  explanation, 
the  relations  which  exist  between  the  condition  of  the  patient 
and  the  virulence  of  the  intestinal  bacteriophage  against  this 
pathogenic  bacterium.  The  upper  tracing  gives  the  number  of 
stools  in  24  hours;  the  single  line  indicating  stools  without  blood, 
the  double  line  those  containing  blood  and  mucus.  On  the  lower 
portion  of  the  chart  is  indicated  (1)  by  the  dotted  line,  the  viru- 
lence of  the  bacteriophage  for  the  colon  bacillus;  (2)  by  the  broken 
line,  the  virulence  of  the  bacteriophage  for  the  stock  strain  of  the 
Shiga  bacillus  which  had  been  maintained  for  a  long  time  under 
laboratory  cultivation;  and  (3)  by  the  heavy  line,  the  virulence 
for  the  Shiga  strain  taken  from  the  patient  himself. 

The  five  cases  given  as  examples  have  been  treated  at  the 
Pasteur  Hospital.  It  has  thus  been  possible  to  follow  them  with 
all  necessary  attention  and  to  obtain  material  for  examination 
as  often  as  the  investigation  demanded;  at  least  once,  often 
several  times,  during  the  course  of  each  day.1 

For  these  examples,  cases  of  different  severity  have  been  selected. 
In  all  of  them  B.  dysenteriae  Shiga  was  isolated  from  the  stools 
at  the  beginning  of  the  disease. 

1.  Germaine  Mel.  .  .  .  (sixteen  years,  fig.  1).  This  was  a 
mild  case  of  dysentery.  The  patient  was  an  inmate  in  an  institu- 
tion where  there  were  about  thirty  young  girls.  During  the 
period  from  the  12th  to  the  22nd  of  July  about  twenty  of  these 
girls  presented  intestinal  disturbances  of  sudden  onset,  accom- 
panied by  a  profuse  diarrhea,  followed  by  a  rapid  amelioration 

1 1  must  here  thank  the  sisters,  nurses  in  the  Pasteur  Hospital,  who 
have  with  unwearying  kindness  provided  me  with  the  numerous  specimens 
which  have  allowed  me  to  follow  the  condition  of  the  patients. 


THE   BACTERIOPHAGE   IN  DISEASE 


177 


of  symptoms.  Within  one  or  two  days  after  the  onset  all  had 
again  become  normal.  In  only  one  or  two  cases  did  the  stools 
contain  traces  of  blood.  In  order  to  establish  a  diagnosis  the 
directrix  was  asked  to  send  a  patient  to  the  Hospital  during  the 
earliest  symptoms. 

Germaine  Mel.  .  .  .  entered  the  Hospital  on  the  18th  of  July. 
From  the  first  stool  passed  after  her  arrival  a  bacillus  presenting 
the  biochemical  characteristics  of  the  Shiga  bacillus  was  isolated 

Day  of  the  Disease 


eo 


FIG.  1.    GERMAINE  MEL.  .  .  .    DYSENTERY  (SHIGA) 


B.  dysenteriae  from  the  patient- 
Virulence  for  "I  B.  dysenteriae,  stock  strain  — 
B.  coli. . 


after  considerable  difficulty.  It  was  inagglutinable,  and  it  was 
only  after  three  passages  on  agar  that  agglutination  was  secured 
(1:500). 

As  can  be  seen  in  the  tracings,  the  number  of  fluid  stools, 
seventeen  on  the  first  day,  fell  quickly  during  the  second  day 
to  two,  without  medication. 

The  intestinal  bacteriophage,  isolated  from  the  fifth  stool  of  the 
first  day,  was  endowed  with  an  extreme  virulence  for  the  bacillus 


178 


THE   BACTERIOPHAGE 


of  the  infection,  and  with  a  somewhat  lower  grade  of  virulence 
for  the  stock  Shiga  strain  and  for  B.  coli. 

The  stools  of  eleven  of  the  inmates  of  this  institution  were 
examined.  Among  the  number  were  nine  who  had  shown  in- 
testinal disturbances  two  or  three  days  previously.  Two  had 
shown  no  morbid  symptoms.  All  of  those  examined  contained 
a  bacteriophage  with  a  high  or  extreme  virulence  for  the  Shiga 
strain  isolated  from  the  stool  of  Germaine  Mel.  ...  as  well  as 
for  the  stock  strain  of  Shiga  and  for  B.  coli. 

Day  of  the  Disease 


'  —  —  -  JN  umber  01  stools 
+  per  24  hours 

+  + 

'• 

. 

9 

o 

\ 

1   16  II  U 

i?  (x  iri  it  4  <j  f  |t0 

^( 

il 

is  IK 

ir 

u 

<r 

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Jo 

Jl 

11 

Jj 

1) 

IS 

«jr 

11  HI 

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—  •* 

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+  + 
+  + 

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f 

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---                   ~!~! 

--- 

FIG.  2.    MARIE  LEB (26  years)      DYSENTERY  (SHIGA) 

!B.  dys enter iae  from  the  patient 
B.  dysenteriae,  stock  strain - 
B.  coli 

-Stools  contained  blood 

Therefore,  with  Germaine  Mel.  .  .  .  there  was  a  bacteriophage 
of  maximum  activity,  even  from  the  beginning  of  the  disease. 
Recovery  took  place  within  twenty-four  hours. 

2.  Marie  Leb (twenty-six  years,  fig.  2).  This  case  was 

one  with  a  mild  dysentery,  due  to  B.  dysenteriae  Shiga.  The 
stools  were  typical,  containing  blood  and  mucus.  Entrance 
to  the  Hospital  took  place  on  the  eighth  day  of  the  disease. 
The  first  stool  containing  blood  had  been  passed  the  day  before. 

Upon  entrance  to  the  Hospital  the  feces  contained  a  bacterio- 


THE   BACTERIOPHAGE   IN   DISEASE 


179 


phage  active  for  the  Shiga  organism  (+),  extremely  active  for 
B.  coli  (+  +  +  +),  and  but  very  slightly  active  for  the  dysentery 
bacillus  found  in  the  patient  f-f ).  Against  this  last  bacillus  the 
virulence  increased  during  the  course  of  the  three  following  days, 
reached  its  maximum  activity  (+  +  ++),  fell  away  somewhat 
(++),  and  then  definitely  regained  its  full  virulence  (+  +  +  +)• 
These  fluctuations  in  virulence  were  reflected  in  the  condition 
of  the  patient.  At  the  end  of  convalescence  there  remained  only 
a  slight  activity  (+)  of  the  bacteriophage  against  B.  coli. 

Day  of  the  Disease 


^2     )H 


;> 

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\\ 

[ 

b 

I 

I 

q 

0 

II 

1 

1 

\ 

i£ 

U!| 

jjj 

m 

ti 

u 

w 

14 

tr 

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11 

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« 

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20 

1 

',      - 

^ 

S^ 

s 

j 

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— 

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f 

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-j 

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- 

! 

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i 
i 

FIG.  3.    VICTOR  KER (6  years)      DYSENTERY  (SHIGA) 

f  B.  dysenteriae  from  the  patient 

Virulence  for  \  B.  dysenteriae,  stock  strain -  - 

(B.coli 

Stools  contained  blood 


3.  Victor  Ker.  ...  (5  years,  fig.  3).  The  dysentery  was  due  to 
the  Shiga  bacillus,  was  of  moderate  severity,  and  was  contracted 
by  contact  with  the  patient  next  discussed.  When  admitted  to 
the  Hospital,  on  the  third  day  of  the  disease,  the  intestinal  bac- 
teriophage already  manifested  an  average  virulence  (++)  for 
the  stock  Shiga  strain  as  well  as  for  the  strain  isolated  from  the 
patient.  This  virulence  increased  rapidly  and  maintained  a 
high  value  up  to  the  time  of  complete  convalescence  (+H — h). 
It  then  abruptly  disappeared. 


180 


THE   BACTERIOPHAGE 


Day  of  the  Disease 


1 


t- 

|S 

\ 

*  J 

\ 

r 

^ 

^ 

s 

<l 

9 

*s_ 

- 

^ 

s 

x. 

u 

o 

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.... 

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,: 

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I 

•^ 

: 

) 

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1 

FIG.  4.    JEAN  KER (6  years)     DYSENTERY  (SHIGA) 

{B.  dysenteriae  from  the  patient 
B.  dysenteriae,  stock  strain 
B.  coli 

Stools  contained  blood 

4.  Jean  Ker.  .  .  .  (six  years,  fig.  4).  This  patient  was  a 
brother  of  the  foregoing.  The  general  condition  was  poor  when 
admitted  to  the  Hospital  on  the  third  day  of  the  disease.  There 
were  from  twenty  to  thirty  bloody  stools  a  day;  a  severe  dysen- 
tery due  to  the  Shiga  bacillus.  On  the  fourth  day  of  the  disease 
there  were  twenty-four  bloody  stools.  The  bacteriophage  was 
feebly  active  (+)  for  B.  coli  and  was  inactive  for  the  Shiga 
bacillus.  The  record  shows  the  following: 


VIRULENCE  OF  THE  BACTERIOPHAGE  AGAINST 

DAT  OF 
DISEASE 

NUMBER  OF  BLOODT  STOOLS 

B.  dysenteriae 
(patient) 

B.  dysenteriae 
(stock) 

B.  ccli 

5th 

23 

0 

+ 

4. 

6th 

13 

0 

_]_^_  _!__!_ 

+  + 

7th 

9 

0 

+4-4- 

+  +  +  + 

8th 

12 

0 

+4- 

+  +  +  + 

9th 

11 

0 

+ 

4.4.4.4- 

10th 

12 

-f 

_l_++4 

4-4-+ 

llth 

12 

+++ 

4-4-4-4- 

+++ 

12th 

(4  of  6  stools  without 

++  + 

_l_4-_l_4- 

+ 

blood) 

THE   BACTERIOPHAGE  IN   DISEASE  181 

From  this  time  on  improvement  became  more  and  more  marked. 
The  activity  of  the  bacteriophage  did  not  disappear  after  con- 
valescence had  been  established. 

In  the  first  three  of  these  cases  the  dysentery  was  mild.  The 
bacteriophage  was  active  at  the  onset,  the  bacterium  did  not 
acquire  a  resistance,  and  its  growth  was  quickly  suppressed.  In 
the  last  case  there  was  a  struggle  and  the  bacillus  acquired  a  resist- 
ance which  was  finally  overcome.  The  condition  of  this  patient 
was  much  more  serious. 

5.  Lans.  .  .  .  (seventy  years,  fig.  5).  This  case  illustrates 
an  extremely  severe  dysentery  due  to  the  Shiga  bacillus.  The 
patient  entered  the  Hospital  on  the  second  day  of  the  disease. 

In  this  case  the  struggle  was  prolonged,  with  fluctuations  due 
to  the  mixed  cultures  formed  in  the  intestine.  The  condition 
of  the  patient  registered  faithfully  the  changes  in  the  struggle. 
It  may  be  noted  particularly  that  the  bacteriophage  manifests 
a  transitory  activity  on  the  eleventh  day  of  the  disease  and  the 
stools  temporarily  lose  their  bloody  character.  But  the  bacillus 
increases  its  resistance  and  this  permits  it  to  develop,  and  blood 
reappears  in  the  stools.  The  disease  is  only  definitely  overcome 
at  a  time  when  the  virulence  of  the  bacteriophage  is  sufficiently 
high  to  dominate  the  resistance  of  the  bacterium. 

Aside  from  the  five  cases  cited  as  examples  others  have  been 
followed,  both  in  France  and  in  Indo-China.  Seventeen  other 
cases  differing  in  severity  were  examined  daily,  and  twenty-nine 
more  were  observed  less  frequently.  In  all  of  the  cases  the  activity 
of  the  bacteriophage  was  manifested  in  an  identical  manner: 

1.  In  case  of  recovery,  the  virulence  of  the  bacteriophage  com- 
mences to  manifest  itself  in  a  marked  manner  toward  B.  coli. 

2.  The  virulence  next  extends  to  the  type  strain  of  the  Shiga 
bacillus,  that  is  to  say,  toward  a  strain  which  has  been  for  a  long 
time  under  artificial  cultivation  and  which,  for  this  reason,  has 
been  deprived  of  much  of  its  resistance. 

3.  It  manifests  itself  next,  more  or  less  quickly,  toward  the 
Shiga  bacillus  isolated  from  the  patient  himself  at  the  onset  of 
the  disease.2 

2  Obviously  it  is  necessary  to  preserve  this  strain  without  replanting. 
The  isolated  colonies  obtained  on  the  original  plates  are  planted  on  several 


182 


THE  BACTERIOPHAGE 


Day  of  the  Disease 

£_j                      ;       II    •     1 

FIG  5.  LANS  (70  years)  DYSENTERY  (SHIGA) 

f  71  Jvewtvrlno  (rn-n-i  fVia  T->oliVnt 

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THE  BACTERIOPHAGE  IN  DISEASE  183 

4.  In  all  cases  the  fluctuations  in  the  virulence,  as  well  as  the 
fluctuations  in  the  resistance  of  the  bacteria,  parallel  the  state 
of  the  patient,  and  the  onset  of  improvement  coincides  with  the 
moment  when  the  virulence  of  the  bacteriophage  dominates 
clearly  the  resistance  of  the  bacterium.  We  thus  see  reproduced 
in  vivo  the  same  mode  of  action  as  that  observed  in  vitro;  per- 
manent and  complete  lysis,  mixed  cultures  with  negative  trans- 
fers, mixed  cultures  with  alternations  in  the  dominating  force. 

In  Indo-China  an  opportunity  was  afforded  to  follow  four  fatal 
cases  of  bacillary  dysentery  in  natives.  At  no  time  during  the 
course  of  the  infection  did  the  intestinal  bacteriophage  show  a 
trace  of  activity  for  the  Shiga  bacillus,  either  for  the  stock  strain 
or  for  that  isolated  from  the  stools  of  the  patients. 

A  last  case  offers  an  especial  interest,  for  it  shows  that  it  is 
not  only  in  vitro  that  the  bacteria  are  able  to  become  refractory 
to  the  action  of  the  bacteriophage.  Although  this  may  occur 
in  vivo  these  cases  must  be  very  rare,  even  exceptional. 

Alix  Desp.  .  .  .  (fifty-six  years).  The  patient  entered  the 
Pasteur  Hospital  on  September  26,  1919.  At  the  time  of  ad- 
mission there  was  a  profuse  mucous  diarrhea  with  thirty  to  forty 
stools  a  day.  Examination  of  the  intestinal  contents  gave  an 
almost  pure  culture  of  a  dysentery  bacillus  presenting  atypical 
characters,  as  follows: 

Non-motile  bacillus.  Gram  negative.  Indol  positive.  No  black- 
ening of  lead  acetate  agar.  No  change  in  neutral  red  media.  Litmus 
sugar  agar  media  not  fermented  with  any  of  the  sugars.  In  Barsiekow's 
medium,  maltose  and  lactose  are  unchanged,  glucose  and  mannite  are 
turned  red.  After  six  transfers  on  agar  it  agglutinated  to  the  titre  (1 :6000) 
with  a  Hiss  agglutinating  serum,  to  1:400  with  an  anti-Flexner  serum  of 
which  the  titre  was  1:6000,  and  was  not  agglutinated  at  1:20  by  an  anti- 
Shiga  serum. 

In  spite  of  these  atypical  characters  it  is,  then,  a  Hiss  strain 
possessing  weak  fermentative  properties. 

When  secured  from  the  body  this  bacillus  was  not  affected  by 
a  bacteriophage  very  virulent  for  a  normal  Hiss  bacillus,  but  it 

agar  tubes  and  a  portion  is  taken  from  these  tubes  for  the  tests  conducted 
during  the  course  of  the  disease.  It  is  well-known  that  resistance  is 
attenuated  by  successive  transplantations. 


184  THE   BACTEEIOPHAGE 

was  lysed  after  about  a  dozen  transplantations.  It  was,  then,  a 
bacillus  which  was  refractory  to  the  bacteriophage  when  taken 
from  the  body. 

At  the  same  time  a  strain  of  bacteriophage  was  isolated  from 
the  stools  of  the  patient.  This  presented  the  following  virulences: 
Shiga  0,  Flexner  +  +  ;  stock  culture  strain  of  Hiss  +  +  +  +  ; 
B.  coli  + ;  the  Hiss  strain  from  the  patient  +.  After  twelve  sub- 
cultures of  the  Hiss  strain  from  the  patient  the  virulence  of  the 
filtrate  was  again  tested.  Perfect  lysis  was  secured,  showing 
that  the  bacillus  had  lost  its  resistance  by  transfers  on  agar. 

The  bacteriophage  of  the  patient  is  active  to  a  maximum  degree 
against  a  stock  strain  of  the  Hiss  bacillus  but  it  is  only  slightly 
active  for  the  individual  strain  causing  the  infection,  with  which 
it  forms,  in  vitro,  mixed  cultures  indefinitely  cultivable.  There 
was  likewise  in  the  intestine  of  the  patient  a  mixed  culture  of 
the  bacteriophage  and  the  refractory  Hiss  strain. 

In  spite  of  every  care  and  repeated  injections  of  anti-dysentery 
serum  the  patient  became  more  and  more  weak;  the  temperature 
oscillated  between  38°  in  the  morning  and  40°C.  in  the  evening; 
the  number  of  stools  gradually  increased  and  became  uncountable 
on  about  the  thirtieth  day;  and  at  about  this  time  the  patient 
fell  into  a  marasmic  condition,  the  temperature  stayed  at  about 
38°C.  and  death  occurred  on  the  thirty-fifth  day. 

Bacteriologically,  the  stools,  tested  each  day,  showed  an  al- 
most constant  bacterial  flora.  The  pathogenic  bacillus  was  always 
abundant,  often  in  almost  pure  culture,  and  presented  the  char- 
acteristics described.  The  virulence  of  the  bacteriophage  in- 
creased continuously  until  the  fifteenth  day  when  it  became  fixed, 
showing: — Shiga  +  +  +  +  ;  Flexner  +  +  +  +  ;  Hiss  +  +  ++, 
B.  typhosus  +  +  +  ;  B.  paratyphosus  A  +  +  +  ;  B.  paratyphosus  B 
+  +  +  ;  B.  coli  +  +  +  +  ;  bacillus  of  the  patient  0  (completely 
refractory)  when  freshly  isolated,  +  -f-  +  after  fifteen  transplants. 

At  autopsy3  there  was  isolated  from  the  contents  of  the  colon, 
from  a  fragment  of  mucous  ulceration,  from  the  liver,  from  the 
spleen,  and  from  the  heart  blood,  a  Hiss  dysentery  bacillus,  pre- 
senting the  same  characteristics  as  that  which  had  been  isolated 

3  Performed  by  L.  Ge"ry,  whom  I  thank  for  the  specimens  he  was  kind 
enough  to  send  me. 


THE   BACTERIOPHAGE   IN  DISEASE  185 

at  the  beginning  of  the  disease .  From  all  the  organs  a  bacteriophage 
was  isolated  presenting  the  same  characters  as  that  which  had  been 
isolated  from  the  stools  and  whose  virulence  has  been  indicated. 

This  case,  altogether  exceptional  (I  believe  that  it  is  the  first 
case  reported  of  a  B.  dysenteriae  Hiss  septicemia)  is  very  interest- 
ing for  it  shows  in  an  unquestioned  manner  the  role  that  the  bac- 
teriophage plays  in  the  defense  of  the  organism.  In  all  of  the 
cases  examined  heretofore  we  have  seen,  either  recovery  starting 
from  the  time  when  the  bacteriophage  had  acquired  sufficient 
virulence  to  dominate  the  pathogenic  bacillus,  or  death  in  the 
case  of  the  failure  of  adaptation.  In  this  last  case,  the  bacteria 
developed  a  refractory  condition,  the  bacteriophage  was  overcome 
and  remained  without  action  whatever  its  virulence  may  have 
been.  The  barrier  thus  being  lacking,  the  bacteria  developed 
freely  and  invaded  the  entire  organism.  The  patient  succumbed 
to  a  septicemia  with  the  Hiss  bacillus. 

This  exceptional  case  provides  us  with  new  information.  A 
bacterium  is  pathogenic  for  a  given  organism  if  it  secretes  sub- 
stances toxic  for  the  cells  of  this  organism.  It  is  the  more  viru- 
lent the  more  capable  it  is  of  development  at  the  expense  of  this 
organism.  The  dysentery  bacilli  are  pathogens  because  of  this 
secretion  of  toxic  substances,  for  they  do  not  invade  the  organism, 
but  remain  localized  in  the  intestine  and  in  the  intestinal  mucosa. 
Nevertheless,  in  the  case  of  the  woman  Desp.  .  .  .  the  Hiss 
strain  was  accidentally  endowed  with  an  extreme  virulence, 
and  this  solely  because  the  bacteriophage  had  been  overcome. 
This  suggests  an  idea  which  we  will  have  occasion  to  confirm  in 
the  following  chapters, — that  the  virulence  of  a  bacterium  at 
any  given  moment  is  the  greater  if  its  resistance  to  the  bacterio- 
phage is  at  this  time  high. 

The  case  Desp.  ...  is  exceptional.  As  a  general  rule  death 
occurs  in  dysentery,  not  because  of  the  acquisition  by  the  bac- 
terium of  a  refractory  condition,  but  by  a  failure  of  the  bacterio- 
phage to  adapt  itself  to  bacteriophagy  toward  the  pathogenic 
organism.  In  the  four  cases  mentioned  above  which  were  fatal, 
a  bacteriophage  active  forlthe  Shiga  bacillus  could  not  be 
isolated  at  any  period  of  the  disease. 


186  THE   BACTERIOPHAGE 

During  the  course  of  the  epidemic  of  dysentery  which  occurred 
in  the  region  of  Paris  during  the  early  autumn  of  1918,  an  oppor- 
tunity was  given  to  observe  twenty-nine  cases  of  benign  diarrhea. 
In  all  of  these  cases  a  bacteriophage  of  very  high  or  extreme  ac- 
tivity for  the  Shiga  bacillus  was  isolated  from  stools  taken  the 
day  after  the  malaise.  This  bacillus  was  the  cause  of  all  the 
severe  cases  studied  at  this  same  time. 

Living  at  this  time  in  a  locality  (Meulan)  where  several  severe 
cases  of  dysentery  were  noted  together  with  a  large  number  of 
cases  of  transitory  diarrhea,  I  examined  the  stools  of  nine  per- 
sons who  were  healthy,  but  who  lived  in  contact  with  individuals 
who  had  had  dysentery.  From  these  nine  individuals  a  bacterio- 
phage of  average  or  high  activity  for  the  Shiga  bacillus  was  iso- 
lated. We  have  noted  above  that  the  same  fact  was  observed 
in  the  institution  where  Germaine  Mel.  .  .  .  had  contracted 
dysentery.  Individuals  who  are  exposed  to  infection  and  who 
resist  show  therefore  in  their  intestine  a  bacteriophage  virulent 
for  the  causative  pathogenic  bacillus,  exactly  like  the  affected 
individuals  who  recover. 

As  a  result,  in  an  epidemic  period  the  simple  cases  of  diarrhea 
must  in  reality  be  cases  of  aborted  bacillary  dysentery,  thanks 
to  the  rapidity  with  which  the  intestinal  bacteriophage  adapts 
itself  to  bacteriophagy  against  pathogenic  bacteria.  And  healthy 
individuals,  living  in  contact  with  affected  people,  are  only  spared 
by  virtue  of  a  still  more  rapid  adaptation  occurring  before  mor- 
bid symptoms  appear.  We  will  find  comparable  facts  in  all  the 
diseases  which  we  will  discuss. 

To  summarize :  the  pathogenesis  and  the  pathology  of  bacillary 
dysentery  are  dominated  by  two  factors,  operating  in  different 
directions;  the  dysentery  bacillus  as  the  pathogenic  agent  and 
the  bacteriophage  as  the  agent  of  immunity.  The  history  of  a 
case  of  dysentery  is  only  the  story  of  the  struggle,  occurring  with- 
in the  body,  between  these  two  factors,  and  the  condition  of  the 
patient  faithfully  reflects  the  vicissitudes  of  the  struggle. 

In  case  of  a  rapid  enhancement  in  the  virulence  of  the  intestinal 
bacteriophage  toward  a  pathogenic  bacillus,  the  latter  is  unable 
to  develop  a  resistance  and  is  destroyed  in  the  struggle,  so  that 
the  disease  aborts  before  the  appearance  of  any  symptoms  or 
manifests  itself  only  in  a  transitory  disturbance. 


THE   BACTERIOPHAGE   IN   DISEASE  187 

The  increase  in  the  virulence  of  the  bacteriophage  for  the  in- 
vading bacterium  may  be  retarded  for  one  of  two  reasons : — First, 
as  a  result  of  unfavorable  intestinal  conditions.  (We  have  seen 
the  considerable  importance,  in  vitro,  of  very  slight  variations 
in  the  reaction  of  the  medium  on  the  development  of  the  ultra- 
microbial  bacteriophage.)  In  accordance  with  the  chemical  and 
physical  state  of  the  intestinal  contents,  one  bacterium  is  favored 
at  the  expense  of  another;  the  intestinal  fermentations,  and  as  a 
result,  the  reaction  of  the  medium  will  vary  according  to  the 
predominating  flora.  The  development  of  the  bacteriophage  is 
then  doubly  influenced,  first,  by  a  change  in  the  state  of  the  medium 
itself,  and  second,  by  changes  in  the  flora  which  increase  or  de- 
crease, according  to  circumstances,  the  bacterial  species  at  the 
expense  of  which  it  normally  develops.  This  of  course,  necessi- 
tates variations  in  virulence  in  response  to  the  variation  in  the 
bacterial  species.  Moreover,  it  has  been  known  for  a  long  time 
that  catarrhal  diarrhea  affects  (provoked  by  the  ingestion  of  un- 
digestible  foodstuffs,  of  green  fruits  in  particular,  or  by  the  "froid 
au  ventre"  so  common  in  tropical  countries)  the  incidence  of 
certain  intestinal  diseases — dysentery  and  cholera  among  others. 

Second,  as  a  result  of  a  more  or  less  marked  degree  of  resistance 
to  the  bacteriophage  of  the  invading  bacillus.  We  have  seen 
that  in  the  course  of  the  disease  the  pathogenic  agent  defends 
itself.  Such  a  bacillus  in  a  state  of  resistance,  ingested  by  a 
healthy  person  will  develop  in  spite  of  the  presence  of  a  bacterio- 
phage, particularly  if  the  latter  is  but  slightly  active,  whereas  a 
non-resistant  bacillus  is  destroyed  without  a  struggle. 

In  cases  of  bacillary  dysentery,  even  very  severe,  but  in  which 
the  patient  improves  rapidly,  the  bacteriophage  manifests  its 
presence  in  a  very  active  manner  at  the  outset,  not  only  for  labora- 
tory strains  of  the  bacillus,  but  for  the  strain  secured  from  the 
patient  himself,  and  this  takes  place  at  the  moment  when  the 
symptoms  begin  to  improve.  There  may  be  a  rapid  increase  in 
the  virulence  of  the  bacteriophage  without  a  corresponding  resist- 
ance in  the  bacterium. 

In  cases  where  the  disease  is  prolonged,  two  cases  may  be 
considered : 


188  THE   BACTERIOPHAGE 

1.  The  bacteriophage  shows  no,  or  but  slight,  activity  as  long 
as  the  condition  of  the  patient  remains  stationary.     The  improve- 
ment occurs  when  the  activity   of  the  bacteriophage  manifests 
itself  in  an  energetic  manner,  not  only  for  the  stock  cultures  of 
the  bacillus  but  also  for  the  strain  derived   from  the  patient. 
There  has  been  a  delay  in  the  adaptation,  then  a  sudden  acquisi- 
tion of  a  high  virulence.     Recovery  takes  place  promptly,  for  the 
pathogenic  bacterium  is  not  able  to  develop  a  resistance. 

2.  At  a  given  moment  of  the  disease  the  virulence  of  the  bac- 
teriophage manifests  a  more  or  less  energetic  action  on  the  stock 
bacilli,  but  on  the  contrary,  it  is  inappreciable  or  but  very  weak 
on  the  strain  taken  from  the  patient.     Here  there  has  been  a  de- 
lay in  the  adaptation,  since  the  bacteriophage  has  gradually  ac- 
quired virulence  for  the  pathogenic  bacillus,  but  this  has  allowed 
sufficient  time  for  the  creation  of  a  resistant  race  of  the  latter. 
As  a  result  there  is  a  struggle,  and  the  condition  of  the  patient 
reveals  the  fluctuations  of  the  struggle. 

This  conflict  is  particularly  to  be  noted  in  cases  of  long  dura- 
tion with  a  relapse.  During  the  latter,  especially,  the  virulence 
of  the  bacteriophage  shows  daily  fluctuations.  At  certain  times 
it  may  be  extreme  for  the  stock  culture,  although  uniformly  very 
weak  for  the  strain  of  the  infection.  Recovery  begins  to  take 
place  at  the  moment  when  the  bacteriophage  shows  an  activity 
as  intense  for  one  strain  as  for  the  other. 

The  disease  has  a  fatal  issue  in  two  cases : 

1.  When  the  bacteriophage  exerts  no  protective  action  through 
a  lack  of  adaptation  to  the  pathogenic  bacillus.    Here  there  is 
no  struggle  at  all,  and  the  bacterium  develops  freely.     In  the 
great  majority  of  such  cases  non-adaptation  is  the  cause  of  death, 
which  then  occurs  quickly. 

2.  In  certain  exceptional  cases  the  pathogenic  bacterium  ac- 
quires an  almost  absolute  resistance, — a  refractory  state.     And 
the  bacteriophage,  whatever  the  degree  of  virulence  it  acquires, 
remains  ineffective.     From  this  moment,  when  the  bacterium 
becomes  equal  to  the  bacteriophage,  the  entire  body  is  invaded 
and  death  ensues  after  a  greater  or  less  length  of  time. 


THE   BACTERIOPHAGE   IN   DISEASE  189 

COLON    BACILLUS    INFECTIONS 

Sometimes  the  colon  bacillus  may  become  pathogenic  and 
may  be  encountered  as  the  etiological  agent  in  diverse  localized 
infections,  or  even  in  septicemias.  It  at  first  appears  strange 
that  so  common  an  organism,  a  normal  inhabitant  of  the  intestine, 
should  at  a  particular  time  develop  pathogenicity.  There  must 
be  "a  something"  which  differentiates  the  pathogenic  B.  coli 
from  the  banal  B.  coli.  It  is  this  which  I  have  tried  to  determine. 

Five  specimens  of  infected  urine  secured  from  individuals 
with  pyelonephritis  have  been  examined.  In  all  of  these  cases 
not  only  was  the  colon  bacillus  present,  but  there  was  a  mixed 
culture  of  B.  coli  and  the  bacteriophage,  as  shown  by  inoculation 
of  the  urine  on  agar.  In  one  of  the  cases  simple  plating  of  the 
urine  on  agar  gave  a  colon  culture  studded  with  plaques,  in  the 
other  four,  agar  cultures  made  after  a  bouillon  growth  gave  the 
same  appearance.  The  colon  bacillus  possessed  a  high  resistance, 
although  it  was  not  entirely  refractory.  Thus  the  struggle  con- 
tinued in  the  organism.  The  ordinary  B.  coli  is  not  pathogenic. 
The  resistant  B.  coli  becomes  so  because  of  its  resistance  to  the 
action  of  the  bacteriophage. 

The  history  of  a  morbid  condition  is  the  history  of  the  struggle 
between  the  bacteriophage  which  attacks  with  its  virulence,  on 
one  side,  and  a  bacterium  susceptible  of  resistance  on  the  other. 
Moreover,  the  struggle  can  be  continued  as  long  as  the  bacterium 
secretes  products  toxic  for  the  infected  body,  but  in  the  last 
analysis,  it  is  the  issue  of  this  conflict  which  decides  the  fate  of  the 
individual. 

We  will  have  occasion  to  return  to  the  case  of  pyelonephritis 


TYPHOID  FEVER  AND  THE  PARATYPHOID  FEVERS 

Several  cases  of  typhoid  fever  of  varied  severity  have  been 
studied  by  the  same  method  as  that  employed  in  bacillary  dys- 
entery. Fourteen  of  these  were  in  the  Pasteur  Hospital  for 
treatment,  and  of  these  the  stools  were  examined  at  least  once 
a  day  throughout  the  course  of  the  disease  and  in  convalescence. 
Fourteen  more  under  treatment  in  other  hospitals  were  followed 


190 


THE   BACTERIOPHAGE 


with  somewhat  fewer  examinations.  In  all  the  charts  which 
follow,  the  following  data  is  presented; — in  the  upper  portion  is 
the  curve  showing  the  temperature ;  in  the  lower  portion  there  are 
three  tracings,  (1)  in  dotted  line,  showing  the  curve  of  the  virulence 
of  the  bacteriophage  for  B.  coli,  (2)  in  broken  line,  showing  the 
virulence  of  the  bacteriophage  for  an  old  laboratory  strain  of 
B.  typhosus,  a  strain  which  has  undergone  a  great  many  transfers 
on  laboratory  media  (this  same  strain  was  used  in  all  the  cases 
studied),  and  (3)  in  solid  line,  indicating  the  curve  of  virulence 

Day  of  the  Disease 


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FIG.  6.    MARDE  Mo (55  years)     CLINICALLY,  TYPHOID  FEVER 


Virulence  for 


B.  typhosus 
B.  coli. . 


B.  paratyphosus  A  — • 

B.  paratyphosus  B  - 

B.  dysenteriae  Shiga 


of  the  bacteriophage  for  the  strain  of  B.  typhosus  from  the  patient 
himself,  isolated  either  by  stool  culture  or  by  blood  culture. 

In  order  to  use  bacilli  as  comparable  as  possible  with  those 
found  in  the  body  of  the  patient  the  strains  were  transplanted 
as  infrequently  as  possible.  In  each  case  an  agar  tube  was  inocu- 
lated with  a  colony  taken  from  the  primary  culture,  and  each 
time  that  a  fresh  culture  was  needed  for  the  preparation  of  sus- 
pensions against  which  the  nitrates  containing  the  bacteriophage 
from  the  patient  were  to  be  tested,  it  was  always  taken  from  this 
tube.  In  this  way,  the  bacteriophage  throughout  the  course 


THE   BACTERIOPHAGE   IN   DISEASE 


191 


of  the  disease  was  tested  against  a  culture  as  nearly  constant  as 
possible,  uniform  especially  from  the  point  of  view  of  the 
resistance  of  the  bacterium. 

For  the  first  three  curves  only  (figures  6,  7,  and  8)  the  organism 
of  the  patients  had  not  been  isolated  (they  had  fevers  which 
appeared  benign)  and  the  curves  of  the  virulence  of  the  bacterio- 
phage  against  the  bacillus  of  the  patient  is,  of  course,  lacking. 
For  these  three  cases  the  virulence  of  the  bacteriophage  against 

Day  of  the  Disease 


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FIG.  7.    Louis  Pi (17  years)     CLINICALLY,  TYPHOID  FEVER 


Virulence  for 


B.  typhosus 

B.  paratyphosus  A  — 

B.  coli 

B.  paratyphosus  B  — 
B.  dysenteriae  Shiga  . 


a  Shiga  dysentery  strain,  and  against  the  paratyphoids  A  and  B 
are  given. 
We  will  select  as  examples  cases  of  different  severity. 

1.  Mild   infections 

These  were  cases  of  typhoid  fever  or  paratyphoid  fever  with  a 
mild  course.  Clinically  they  were  typhoid  fever  but  the  blood 
and  stool  cultures  were  negative.  The  curves  for  these  three 
cases  are  given  on  pages  190,  191  and  192. 


192 


THE   BACTERIOPHAGE 


1.  Marie  Mo.  .  .  .  (fifty-five  years,  fig.  6). 

2.  Louis  Pi.  ...  (seventeen  years,  fig.  7). 

3.  Franc. ois  Jod.  .  .  .  (thirty-four  years,  fig.  8). 

In  these  cases  the  virulence  of  the  intestinal  bacteriophage 
was  determined  for  B.  coli,  B.  typhosus,  B.  paratyphosus  A  and 
Bt  and  B.  dysenteriae  Shiga.  It  is  needless  to  comment  on  these 
observations,  since  examination  of  the  curves  is  more  instructive 
than  would  be  an  explanation. 

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FIG.  8.    FRANCOIS  JOD (34  years)     CLINICALLY,  TYPHOID  FEVER 


Virulence  for  • 


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coli. . 


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B,  paratyphosus  B  - 
B.  dysenteriae  Shiga 


What  is  the  causative  bacillus  in  each  of  these  three  cases? 
It  is  indeed  difficult  to  make  a  diagnosis  by  means  of  the  bacterio- 
phage, which  as  we  have  seen,  but  rarely  develops  a  single 
virulence. 

This  virulence  extends  to  other  bacteria  of  the  same  group  to 
a  more  or  less  marked  degree,  and  this  fact  is  particularly  in 
evidence  when  working  with  the  representatives  of  the  colon- 
typhoid-paratyphoid-dysentery  group.  It  appears,  however,  in 
the  case  of  Louis  Pi.  ...  that  the  causative  bacillus  must  have 
been  the  typhoid  bacillus,  with  Marie  Mo.  .  .  .  B.  paratyphosus  A, 
and  in  Frangois  Jod.  .  .  .  B.  paratyphosus  B. 


THE    BACTERIOPHAGE   IN    DISEASE  193 

It  should  be  noted  that  in  these  three  cases  in  which  improve- 
ment was  rapid,  the  curves  representing  the  virulence  of  the 
bacteriophage  are  comparable.  It  is  also  to  be  noted  that  in 
all,  the  accessory  virulences  for  B.  dysenteriae  Shiga  and  for  B. 
coli  are  very  high,  and  that  the  acquisition  of  virulence  for 
B.  typhosus  and  B.  paratyphosus  A  and  B  is  early  and  is  maintained 
up  to  the  beginning  of  convalescence.  In  the  case  of  Louis  Pi.  . 
the  abrupt  deflection  in  virulence  on  the  twenty-second  day 
preceded  a  slight  relapse  which  occurred  on  the  twenty-fifth  to 
the  thirty-second  day.  This  complication  did  not  prove  serious 
since  the  virulence  of  the  bacteriophage  increased  gradually 
from  the  twenty-third  day. 

2.  Severe  injections 

The  three  cases  cited  here  were  serious,  with  both  stool  and  blood 
cultures  positive.  All  were  infected  with  B.  typhosus. 

1.  Renee  Mar.  .  .  .  (thirty-two  years,  fig.  9). 

The  bacteriophage  was  from  the  beginning  virulent  for  B.  coli, 
and  remained  so  during  the  course  of  the  disease,  throughout 
convalescence,  and  up  to  the  time  when  the  patient  was  dis- 
charged from  the  hospital  completely  cured.  It  may  be  noted 
that  the  acquisition  of  virulence  by  the  bacteriophage  for  the 
bacillus  of  the  infection  coincides  with  the  first  defervescence. 
Then  this  virulence  became  reduced  and  the  temperature  again 
went  up.  The  infection  is  definitely  overcome  at  the  time  when 
this  virulence  is  again  established. 

2.  Juliette  Ou.  .  .  .  (thirty-six  years,   fig.   10). 

3.  Jeanne  Del.  .  .  .  (twenty  years,  fig.  11). 

The  curves  for  these  two  cases  are  self-explanatory. 

8.  Typhoid  fever  with  relapse 

1.  GilberteFon.  .  .  .  (four  years,  fig.  12). 

On  the  sixteenth  day  the  disease  appeared  ended.  However, 
the  virulence  of  the  bacteriophage  toward  the  bacillus  of  the 
patient  disappeared  before  the  end  of  the  crisis  and  the  destruc- 
tion of  the  pathogenic  bacteria  was  not  complete.  Whereupon 
there  was  a  relapse,  very  severe,  which  did  not  show  improvement 
until  the  bacteriophage  recuperated  with  a  virulence  sufficient 
to  control  the  resistance  of  the  bacterium. 


194 


THE   BACTERIOPHAGE 


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198  THE  BACTERIOPHAGE 

4.  Typhoid  fever  of  extreme  severity 

1.  Andre"e  Dess.  .  .  .  (thirty  years,  fig.  13). 

2.  Jeanne  Cot.  .  .  .  (twenty-four  years,  fig.  14). 

In  these  two  cases  strains  of  B.  typhosus  were  isolated  at  different 
times  during  the  course  of  the  disease.  These  bacilli  presented 
a  marked  resistance  to  the  action  of  a  very  active  strain  of  anti- 
typhoid bacteriophage  and  lost  this  resistance  only  after  about 
ten  transfers  on  agar.  It  is  to  be  noted  that  at  their  isolation 
from  the  organism  these  bacilli  were  inagglutinable  (this  fact 
has  frequently  been  observed)  and  that  they  did  not  become 
agglutinable  until  after  a  series  of  cultures.  This  transitory 
inagglutinability  is,  as  we  have  seen,  associated  with  resistance 
to  the  action  of  the  bacteriophage. 

Examination  of  the  curves  shows  clearly  the  struggle  which 
was  carried  on  within  the  organism  between  the  bacterium  and 
the  bacteriophage,  and  the  repercussions  of  this  campaign  upon 
the  state  of  the  patient. 

We  find  then,  in  typhoid  fever, — an  intestinal  infection  compli- 
cated by  a  septicemia — the  same  facts  as  seen  in  bacillary 
dysentery. 

The  virulence  of  the  bacteriophagous  ultramicrobe  isolated 
from  the  stools  of  the  typhoid  patient  is  not  limited,  in  general, 
to  a  single  pathogenic  bacillus;  at  one  and  the  same  time  it  ex- 
tends, in  some  degree,  to  some  or  all  of  the  bacilli  of  the  colon- 
typhoid-dysentery  group.  This  fact  is  particularly  noted  in 
mild  cases  or  those  of  average  severity.  In  the  severe  cases  the 
bactericidal  action  is  more  specific  and  is  often  limited  to  the 
specific  pathogenic  organism  and  to  B.  coli,  the  latter  always 
being  attacked.  In  certain  very  severe  cases  the  specificity 
becomes  such  that  up  to  the  beginning  of  actual  improvement 
only  the  bacillus  isolated  from  the  patient  is  attacked,  whether 
it  has  been  secured  by  stool  or  by  blood  culture,  to  the  exclusion 
of  other  bacilli,  taken  either  from  old  laboratory  cultures  or  from 
strains  recently  isolated  from  other  patients.  It  seems,  then, 
that  in  the  course  of  their  struggle  each  of  the  two  organisms 
present, — bacteriophage  and  bacterium — acquires  an  individual 
personality,  which  differentiates  them  from  other  organisms  of 


THE   BACTERIOPHAGE   IN   DISEASE 


199 


ft 


200 


THE   BACTERIOPHAGE 


THE   BACTERIOPHAGE   IN   DISEASE  201 

the  same  species  rendered  banal  as  a  result  of  cultivation.  How- 
ever, this  individuality  is  effaced  by  cultivation,  both  in  the  case 
of  the  bacteriophage,  which,  after  a  few  passages  at  the  expense 
of  the  bacillus  to  which  it  is  sensitive  may  develop  activity  toward 
any  strain  of  B.  typhosus,  and  of  the  typhoid  bacillus  which  is  then 
able  to  be  attacked  by  any  strain  of  antityphoid  bacteriophage. 

Typhoid  fever  is  not  a  purely  intestinal  infection  as  is  dysentery. 
In  the  latter  it  can  be  understood  how,  when  all  of  the  pathogenic 
bacteria  of  the  intestine  or  of  the  mucosa,  that  is,  those  in 
proximity  to  the  ultramicrobes,  have  been  destroyed  the  disease 
ends  ipso  facto.  In  typhoid  fever  there  is  in  addition  a  septicemia 
and  even  though  the  destruction  of  the  bacilli  contained  in  the 
intestinal  contents  is  sufficient  to  delay  the  appearance  of  the 
disease  or  to  restrain  it  from  the  beginning,  it  may  not  be  adequate 
to  overcome  the  infection  once  the  pathogenic  bacilli  have  invaded 
the  organism. 

We  will  see  later  that  the  protective  action  of  the  bacteriophage 
is  not  limited  to  the  intestine.  The  intervention  of  the  bac- 
teriophage, even  in  the  same  organism,  may  manifest  itself  in 
different  ways. 

In  the  chapter  dealing  with  the  properties  of  the  bacteriophage 
we  have  seen  that  the  products  which  it  secretes  are  possessed 
of  an  extremely  high  opsonizing  power.  A  culture  of  an  antity- 
phoid bacteriophage  is  precipitated  by  the  addition  of  four  vol- 
umes of  96  per  cent  alcohol.  The  precipitate  is  allowed  to  remain 
in  contact  with  the  alcohol  for  forty-eight  hours,  a  time  adequate 
to  ensure  the  complete  destruction  of  all  of  the  bacteriophagous 
germs.  One  centigram  of  this  moist  precipitate  is  dissolved  in  ten 
cubic  centimeters  of  saline.  In  determining  the  opsonic  index,  it  will 
be  seen  that  under  the  action  of  the  lysin  the  leucocytes  become  so 
loaded  with  typhoid  bacilli  that  it  is  impossible  to  count  the  num- 
ber of  organisms  ingested.  The  opsonic  index  is  certainly  higher 
than  fifty.  It  is  possible  that  the  lysin  secreted  in  the  intestine 
as  soon  as  the  bacteriophage  has  acquired  a  virulence  to  dissolve 
the  typhoid  bacilli  may  be  resorbed  and  pass  into  the  circulation, 
and  thus  assure  the  destruction  of  the  bacilli  by  phagocytosis. 

On  the  other  hand,  the  bacteriophagous  ultramicrobe  itself 
does  not  remain  strictly  localized  in  the  intestine;  at  times  it 


202  THE   BACTERIOPHAGE 

passes  into  the  circulation.  This  has  not  been  demonstrated  in 
man  for  it  is  not  practicable  to  carry  out  on  man  the  repeated 
blood  examinations  which  such  a  study  requires.4  However,  in 
paratyphoid  fever  in  the  rat  induced  by  the  ingestion  of  a  very 
virulent  strain  of  B.  typhi  murium  a  transitory  appearance  of  the 
ultramicrobe  in  the  blood  has  been  demonstrated  by  cardiac 
puncture  made  between  the  fourth  and  sixth  days  after  the  inges- 
tion of  the  infectious  material.  All  of  the  rats  in  which  this 
phenomenon  occurred  were  protected.  At  this  time  a  bacterio- 
phage  active  for  the  pathogenic  bacillus  was  present  in  the 
intestine  and  the  rats  resisted  infection. 

In  the  third  place  we  will  see  experimentally  that  the  dissolved 
products  found  in  the  cultures  of  the  bacteriophage  provoke, 
after  an  incubation  period,  the  development  of  an  "  organic  im- 
munity" so  potent  that  it  borders  on  a  refractory  state.  These 
dissolved  products  likewise  form  in  the  intestine  of  the  patient, 
and  even  within  the  body,  since  it  is  possible  for  the  bacteriophage 
to  pass  into  the  circulation  in  a  septicemia. 

Twenty-eight  more  non-fatal  cases  of  typhoid  fever  were  studied 
in  order  to  determine  the  influence  of  the  bacteriophage  on  the 
course  of  the  disease.  Three  fatal  cases  were  observed.  In  these 
three  cases,  at  no  period  of  the  disease  could  the  presence  of  a 
bacteriophage  be  demonstrated  active  for  B.  typhosus,  either  for 
a  stock  strain  or  for  the  bacillus  from  the  patient.  Furthermore, 
examination  of  the  strains  from  the  intestinal  contents  from  five 
individuals  who  had  died  of  typhoid  failed  to  show  any  activity 
for  the  typhoid  bacillus.  But  the  bacteriophage  was  not  entirely 
absent,  since  in  six  of  these  eight  cases  a  bacteriophage  of  moder- 
ate activity  for  the  colon  bacillus  was  found.  This  bacteriophage 
did  not,  however,  show  any  activity  for  the  pathogenic  organisms. 
Death  in  typhoid  fever  results,  usually,  because  of  a  failure  of  the 
bacteriophage  to  adapt  itself  for  the  bacteriophagy  of  the  in- 
vading bacillus. 

May  death  occur  because  of  the  acquisition  of  a  resistant  con- 
dition by  the  typhoid  bacillus,  which  protects  it  from  the  action 

4  Beckerich  and  Hauduroy  have  very  recently  found  it  in  blood  cultures. 
It  is  essential  to  examine  them  systematically  for  the  bacteriophage;  the 
negative  cultures  in  particular. 


THE   BACTERIOPHAGE   IN   DISEASE  203 

of  the  bacteriophage,  as  we  have  seen  in  the  case  of  dysentery? 
There  has  been  no  opportunity  to  establish  this  up  to  the  present 
but  it  is  the  more  probable,  since,  in  vitro  as  in  vivo,  the  tendency 
toward  resistance  is  certainly  more  marked  for  the  typhoid  bacillus 
than  for  B.  dysenteriae.  In  any  case,  this  cause  of  death  is  cer- 
tainly the  exception,  even  in  typhoid.  It  must  necessarily 
accompany  a  septicemia  when  it  occurs. 

In  typhoid,  as  in  dysentery,  the  investigation  of  the  virulence 
of  the  bacteriophage  is  of  prognostic  significance.  It  is  sufficient 
to  verify  simultaneously  the  virulence  of  the  intestinal  bacterio- 
phage of  the  patient  toward  B.  coli,  toward  the  pathogenic  bacillus 
taken  from  the  patient,  and  toward  a  stock  culture  of  B.  typhosus. 
A  comparison  of  these  three  results  furnishes  the  information 
desired.  The  detection  of  resistance  in  the  pathogenic  bacterium 
would  indicate  a  poor  prognosis,  and  that  in  proportion  as  the 
resistance  is  the  more  pronounced.  The  establishment  of  a  re- 
fractory state  in  the  bacterium,  resulting  in  the  formation  of  a 
mixed  culture  in  the  intestine  accompanying  a  septicemia,  im- 
plies a  fatal  outcome  with  a  quick  maturity. 

To  summarize:  in  all  of  the  cases  of  typhoid  fever  studied, 
whatever  may  have  been  their  severity,  the  appearance  in  the 
bacteriophagous  ultramicrobe  of  virulence  for  the  pathogenic 
bacillus  has  been  preceded  by  an  increase  in  virulence  for  B.  coli, 
which  has  always  begun  in  the  course  of  the  second  week  and  has 
rapidly  attained  great  intensity.  This  activity  is  maintained 
during  the  entire  course  of  the  infection  and  appreciably  decreases 
only  during  convalescence,  sometimes  even  later.  On  the  con- 
trary, the  development  of  virulence  for  the  pathogenic  bacillus 
has  varied  according  to  the  severity  of  the  disease.  In  cases  that 
were  mild  or  of  average  severity  the  activity  of  the  bacteriophage 
for  this  bacillus  appears  before  the  end  of  the  second  week  and 
disappears  toward  the  end  of  convalescence.  The  activity  for 
B.  coli  and  for  B.  typhosus  is  there  parallel.  In  the  severe  cases 
the  activity  for  the  typhoid  bacillus  only  commences  to  manifest 
itself  in  an  energetic  manner  towards  the  beginning  of  definite 
improvement.  It  persists  for  a  greater  or  less  length  of  time,  in 
some  cases  up  to  the  middle  of  the  period  of  convalescence. 

In  the  forms  with  relapse  and  recrudescence  the  struggle  is 
complicated  by  the  fact  of  the  acquisition  of  a  resistance  by  the 


204  THE  BACTERIOPHAGE 

bacteria,  and  it  is  only  toward  the  decline  of  this  relapse  or  of  the 
recrudescence  that  the  virulence  of  the  bacteriophage  is  sufficient 
to  definitely  control  the  resistance  of  the  bacterium.  Here,  the 
activity  of  the  bacteriophage  is  maintained  up  to  complete  re- 
covery, that  is  to  say,  up  to  the  moment  when,  because  of  a  total 
destruction  of  the  pathogenic  bacteria,  the  ultramicrobe  is  no 
longer  able  to  develop  at  their  expense. 

In  all  cases,  the  condition  of  the  patient  faithfully  registers 
the  vicissitudes  of  the  struggle  taking  place  within  the  body 
between  the  bacteriophage  and  the  invading  bacterium. 

AVIAN    TYPHOSIS 

The   disease 

Avian  typhosis  is  a  disease  affecting  principally  the  Gallinaceae. 
Despite  its  frequency  it  for  a  long  time  remained  undetected, 
confounded  with  chicken  cholera.  This  last  disease  is,  in  reality, 
very  rare.  In  1919,  in  investigating  epizootics  for  the  purpose 
of  testing  on  domestic  animals,  which  allow  of  experimentation, 
the  conclusions  reached  as  to  the  role  of  the  bacteriophage  in 
human  dysentery  and  typhoid,  an  extended  focus  of  "chicken 
cholera"  was  found  in  the  Department  of  the  Aube.  In  the  first 
examinations  the  error  which  had  been  made  became  apparent; 
it  was  the  disease  known  in  the  United  States  as  "fowl  typhoid/ ' 
whose  existence  in  France  had  up  to  that  time  been  unrecognized. 
Shortly  after  this  numerous  foci  throughout  the  surrounding 
territory  were  discovered. 

Fowl  typhoid,  which  will  here  be  called  fowl  typhosis,  is  a  very 
interesting  disease.  Its  study  is  complicated  by  the  existence 
of  several  "  paratyphoses"  which  resemble  still  more  the  human 
typhoid.  The  pathogenic  agent,  B.  gallinarum  Klein,  studied  by 
Moore  under  the  name  of  B.  sanguinarium,  presents,  with  the 
exception  of  motility,  all  of  the  characteristics  of  the  bacillus  of 
Eberth  (B.  typhosus).  It  is  even  agglutinated  to  titre  by  an  anti- 
typhoid serum.  Aside  from  this  type  bacillus  there  are  often 
found,  in  the  same  foci,  bacilli  presenting  different  agglutinative 
and  biochemical  reactions.  The  clinical  type  of  the  infection 
which  they  provoke  does  not  differ  from  that  caused  by  the 


THE   BACTERIOPHAGE   IN   DISEASE  205 

typhoid  type.  These  differing  species  of  bacteria  have  up  to  the 
present  been  studied  only  by  American  workers;  Ph.  Hadley  among 
others,  who  describes  B.  pullorum  A,  B.  pullorum  B,  B.  jeffersonii, 
B.  rettgerei,  and  B.  pfaffi.  A  discussion  of  the  distinctive  char- 
acters of  these  different  bacilli  will  not  be  presented  here  since 
it  would  not  be  germane  to  the  study  with  which  we  are  concerned.5 
It  is  sufficient  to  know  that  in  France  in  the  epizootic  of  1919  the 
most  frequent  pathogenic  agent  was  of  the  B.  gallinarum  type 
(found  in  57  of  73  examinations).  Along  with  B.  gallinarum 
other  forms  have  been  found: — B.  pullorum  A  (once),  B.  pullorum 
B  (6  times),  B.  jeffersonii  (4  times),  and  B.  pfaffi  (4  times).  In 
a  single  focus,  of  which  the  centre  was  found  in  the  village  of 
Trainel  (Aube),  a  paratyphosis  infection  occurred  due  solely  to 
B.  pfaffi  without  admixture  with  bacilli  of  the  true  typhosis  type. 

The  clinical  picture  hardly  varies  whatever  may  be  the  causa- 
tive bacillus.  A  typical  observation  follows. 

On  the  evening  of  May  24  the  chicken  appeared  perfectly  well. 
On  the  morning  of  May  25  it  remained  apathetically  on  the  ground 
of  the  poultry-yard  and  took  no  measures  for  its  defense.  The 
next  day,  toward  noon,  it  appeared  somnolent,  the  plumage 
rough,  the  eyes  half-closed,  the  crest  slightly  violet  colored.  It 
did  not  eat  or  drink,  and  remained  humped  up  "in  a  ball."  The 
inspirations  were  deep,  twenty-five  per  minute.  There  was  a 
greenish  yellow  diarrhea  with  portions  definitely  yellow.  The 
condition  became  worse  in  the  afternoon.  It  fell  on  its  side  at 
about  8  o'clock  and  died  a  few  minutes  later.  The  necropsy 
showed  the  crest  to  be  violet  in  color,  with  spots  of  the  same 
nature  over  the  skin.  The  liver  was  voluminous,  congested,  and 
presented  foci  of  degeneration.  There  was  a  pericarditis. 

By  direct  microscopic  examination  the  blood  at  first  appeared 
negative,  but  a  very  careful  search  revealed  three  bacilli  in  a  whole 
smear.  The  blood  and  tissues  when  cultured  gave  a  pure  growth 
of  B.  gallinarum,  and  this  organism  was  also  found,  very  abun- 
dantly, in  the  intestinal  contents. 

5  Readers  who  are  interested  in  the  subject  will  find  much  useful  informa- 
tion in  the  contribution  by  Ph.  Hadley  "The  Colon-Typhoid  Intermediates 
as  Causative  Agents  oj  Diseases  in  Birds."  Bulletin  No.  174,  Rhode  Island 
Agric.  Exper.  Sta.,  1918. 


206  THE  BACTERIOPHAGE 

Sometimes  death  occurs  more  rapidly  still,  in  certain  cases  in 
a  striking  manner.  Epizootics  of  avian  typhosis  have  a  high 
mortality.  In  1919  foci  existed  throughout  the  extent  of  France. 
In  general,  the  epizootic  begins  quickly;  within  the  space  of  three 
or  four  weeks  a  half,  three-quarters,  sometimes  more,  of  the 
fowls  on  a  farm  succumb.  Then  the  disease  assumes  a  sporadic 
character,  only  an  occasional  animal  dying  during  the  course  of  a 
year.  The  disease  may  disappear  for  a  few  months  and  then  reap- 
pear. The  annual  mortality  amounts  to  forty  to  seventy  per 
cent  of  the  population  of  the  infected  poultry-yards.  Young 
adults  are  the  most  susceptible,  then  the  old  animals;  the  chicks 
are  in  general  spared. 

Epizootics  of  typhosis  extend  rapidly  over  large  areas;  cer- 
tain Departments  were  contaminated  throughout  in  1919.  The 
establishment  of  a  new  focus  begins  by  the  importation  of  the 
organism  from  an  infected  region,  either  through  the  agency  of  a 
flock  of  sheep  or  herd  of  cattle,  or  by  horsemen  (this  last  mode  of 
dissemination  was  particularly  frequent  during  the  war;  this 
explains  the  extension  of  the  disease  during  the  years  1917  and 
1918).  The  disease  rages  for  a  few  days  on  a  farm,  passes  to  a 
neighboring  farm,  and  then  extends  rapidly  into  the  surrounding 
villages. 

The  pathogenic  bacillus  remains  alive  and  virulent  during 
several  months  in  the  regions  where  the  infection  has  been  epi- 
demic. In  several  tests  it  has  been  shown  that  an  isolated  in- 
fected chicken-yard,  cleaned  and  left  unoccupied  for  six  to  eight 
months,  still  contains  virulent  germs,  for,  when  repopulated  with 
chickens  from  a  region  free  of  the  disease,  the  infection  breaks 
out  again  within  a  few  days  among  the  new  occupants. 

Avian  typhosis  being  a  disease  in  general  but  little  known,  I 
have  thought  it  useful  to  consider  it  in  some  detail,  since  it  will 
allow  us  the  better  to  understand  the  facts  now  to  be  presented. 

The  role  of  the  bacteriophage  in  the  course  of  the  disease 

Because  of  the  exceptional  severity  of  the  infection  in  avian 
typhosis  it  has  been  possible  to  follow  only  four  cases  which  re- 
covered. In  all,  the  picture  has  been  identical.  In  the  morn- 
ing the  infected  chicken  remains  on  the  ground,  " balled  up," 


THE   BACTERIOPHAGE   IN   DISEASE  207 

the  feathers  roughened,  and  with  the  characteristic  diarrhea. 
The  appearance  is  the  same  as  in  the  fowls  which  succumb.  At 
this  stage  of  the  infection  examination  of  the  feces  gives  results 
such  as: 

B.  gallinarum,  present  in  abundance. 

Intestinal  bacteriophage,  virulent  for  B.  coli  -f  (in  2  cases) 
or  ++  (in  2  cases);  for  B.  gallinarum  0  (in  the  four  cases).  The 
blood  culture  was  positive  in  the  two  cases  in  which  it  was  done; 
the  blood  for  culture  being  taken  aseptically  by  puncture  of  the 
crest. 

During  the  course  of  the  day  the  condition  remains  the  same 
as  that  shown  by  animals  which  die.  This  state  is  prolonged  and 
the  next  morning  the  chicken  still  appears  the  same.  Examina- 
tion of  the  feces  at  this  time  shows: 

B.  gallinarum  present  in  three  cases,  absent  in  one. 

Intestinal  bacteriophage  virulent  for  B.  coli  +  +  +  (4  cases); 
for  B.  gallinarum  +  (in  3  cases)  +  +  +  (in  1  case).  Towards 
noon,  in  one  case,  in  the  course  of  the  afternoon  in  the  three  others, 
blood  cultures  were  negative.  In  three  eases  a  bacteriophage 
active  for  B.  gallinarum  was  found  in  the  blood.  The  blood  which 
was  ultrasterile  was  that  of  the  chicken  whose  condition  was  the 
best  at  this  time  and  which  had  shown  no  pathogenic  bacilli  in 
the  intestinal  tract  in  the  morning.  The  presence  of  the  bac- 
teriophage in  the  blood  is  extremely  transitory. 

On  the  morning  of  the  third  day  the  animals  appeared  normal, 
they  drank  a  great  deal,  ate  some  grain,  and  the  diarrhea  was 
less  profuse.  Examination  of  the  feces  showed: 

B.  gallinarum  absent  in  the  four  cases. 

Intestinal  bacteriophage  active  for  B.  coli  +  +  +  (4  cases), 
for  B.  gallinarum  +  +  -f-  (3  cases)  +  +  +  +  (1  case).  Blood 
cultures  were  negative:  no  bacilli,  no  ultramicrobes. 

On  the  fourth  day  the  animals  were  practically  normal. 

In  the  four  chickens  which  recovered  the  bacteriophage  re- 
mained active  for  B.  gallinarum  for  a  very  long  time.  After 
three  months  it  showed  the  same  degree  of  activity  as  at  the  time 
of  recovery.  In  one  of  them,  in  which  it  has  been  possible  to 
make  an  examination  after  five  months,  it  was  still  as  active  as 
at  first.  We  will  see,  from  experimental  observations  that  this 


208  THE  BACTEKIOPHAGE 

persistence  of  virulence  depends  solely  upon  the  fact  that  the 
pathogenic  bacillus,  distributed  in  profusion  in  the  exterior  en- 
vironment, is  frequently  ingested  by  the  animal  and  this  maintains 
the  virulence  of  the  intestinal  bacteriophage  since  it  is  able  to 
grow  at  its  expense. 

The  feces  of  about  one  hundred  chickens  which  had  died  of 
avian  typhosis  were  examined.  In  no  case  was  there  a  bacterio- 
phage active  for  B.  gallinarum  or  for  any  of  the  bacillary  agents 
of  the  paratyphoses.  Nevertheless  the  bacteriophage  had  been 
present  for  it  could  be  disclosed  (91  times  in  97  examinations) 
because  of  the  activity  shown  for  one  or  several  species  of  the 
colon-typhoid-dysentery  group.  One  sees  clearly,  then,  that 
the  lack  of  defense  is  not  due  to  the  absence  of  the  bacteriophage, 
but  solely  to  the  fact  that  the  intestinal  bacteriophage  remained 
passive  because  it  failed  to  acquire  a  virulence  for  the  pathogenic 
bacillus. 

To  summarize:  as  in  dysentery  and  in  typhoid  fever  in  human 
beings,  the  acquisition  of  virulence  by  the  intestinal  bacteriophage 
for  the  pathogenic  bacterium  is  the  sine  qua  non  of  recovery. 

Rdle  of  the  bacteriophage  in  the  course  of  the  epizootic 

Because  of  the  dissemination  and  extent  of  the  disease  it  was 
possible  to  study  the  role  of  the  bacteriophage  in  the  course  of  the 
epizootic  as  well  as  in  the  course  of  the  disease  in  the  individual 
infected  animal. 

Let  us  consider  first  a  fact  bearing  on  the  territory  involved  in 
the  epizootic.  During  the  last  three  years  eighty-one  examina- 
tions have  been  made  upon  the  feces  of  barn-yard  animals,  not 
only  in  France  but  also  in  Indo-China,  in  regions  where  avian 
typhosis  had  not  occurred  in  epidemic  form  among  the  fowls  for 
several  years.  In  each  of  these  examinations  a  bacteriophage 
active  for  one  or  several  of  the  bacilli  of  the  colon-typhoid-dysen- 
tery group  was  isolated,  but  in  no  instance  has  the  bacteriophage 
shown  any  detectable  activity  for  B.  gallinarum. 

In  contaminated  regions  the  situation  is  quite  different.  As 
an  example,  observations  made  on  a  farm  located  at  Pougy-sur- 
Aube  may  be  cited,  where  the  disease  was  followed  very  closely. 
The  disease  appeared  in  1917  in  July.  Within  the  period  of  a 


THE   BACTEBIOPHAGE   IN   DISEASE  209 

month  fifty-one  of  the  ninety-eight  fowls  died;  then  the  epizootic 
disappeared.  In  May,  1918  it  reappeared  in  less  violent  form. 
Twenty-five  of  one  hundred  and  four  fowls  died  in  the  period 
from  May  to  September,  and  it  again  disappeared.  In  1919  it 
broke  out  again  early  in  April.  On  the  21st  of  May,  twenty-one 
of  eighty  had  died.  At  this  time  I  began  my  observations. 

On  May  21,  specimens  of  the  excrement  of  thirty  of  the  fifty- 
nine  survivors  were  taken.  Examination,  made  later  in  the  lab- 
oratory, showed  in  twenty-six  a  bacteriophage  of  weak  or  moder- 
ate activity  for  B.  gallinarum  (23  were  +,  3  were  ++),  in  four 
it  was  absent.  On  May  22,  two  chickens  contracted  the  disease. 
The  strains  taken  the  day  before  were  numbered  and  examination 
showed  that  an  active  bacteriophage  had  not  been  found  in  these 
two  animals.  On  May  23  one  of  the  two  chickens  affected  the 
day  before  died.  On  May  24,  a  third  chicken,  sick  in  the  morning, 
died  in  the  following  night.  Its  excrement,  collected  on  May  22, 
did  not  contain  a  bacteriophage  active  for  B.  gallinarum.  On 
the  morning  of  May  24  the  chicken  which  had  been  taken  sick 
on  May  22  and  which  had  resisted  showed  in  its  intestinal  con- 
tents a  bacteriophage  of  extreme  activity  (+  +  ++)  toward  the 
pathogenic  bacillus.  On  May  26  the  fourth  chicken,  one  of  those 
whose  f  eces  had  not  showed  an  active  bacteriophage  when  examined 
on  May  22,  was  affected.  It  resisted,  and  on  May  28  its  symptoms 
had  disappeared.  The  disease  disappeared  suddenly  and  during 
the  next  three  months  no  new  cases  developed. 

On  May  30  the  feces  of  thirty  chickens  were  examined  and  the 
following  results  were  obtained: 

Virulence  for  B.  gallinarum;  in  five  +  +  ++>  in  twenty-one 
+  +  +  ,  in  four  +  +  . 

We  see,  then,  on  May  22,  four  animals  among  thirty  in  which 
the  intestinal  bacteriophage  lacked  activity  for  the  pathogenic 
bacillus.  These  four  animals  contracted  the  disease  during 
the  four  following  days.  In  the  twenty-six  specimens  collected 
on  May  22  and  showing  positive  results,  the  bacteriophage  showed 
a  relatively  weak  virulence.  Nine  days  later  this  activity  was 
very  much  greater,  that  is,  at  the  time  when  the  epizootic  ceased. 
What,  then,  took  place  in  this  interval?  The  bird  which  became 
sick  on  May  22  and  which  resisted  showed  in  its  feces,  when  ex- 


210  THE   BACTERIOPHAGE 

amined  on  May  24,  a  bacteriophage  endowed  with  a  considerable 
activity  for  the  pathogenic  agent. 

Here  is  a  second  example  of  the  same  general  nature,  giving 
the  results  secured  on  farm  M.  .  .  .  at  Vericourt  (Aube).  The 
epizootic  first  appeared  among  the  flock  of  twenty-five  chickens 
in  May,  1919.  The  first  animal  died  on  May  18.  On  the  next 
day  twelve  specimens  of  excreta  were  collected  at  random.  Three 
only  contained  a  bacteriophage,  and  that  of  feeble  activity,  for 
B.  gallinarum.  From  May  19  to  26  twelve  birds  contracted  the 
disease  and  of  these  eleven  died.  One,  which  became  sick  on 
May  23,  showed  on  May  25  a  strongly  active  bacteriophage 
(H-  +  H-  for  B.  gallinarum)  and  recovered.  The  epidemic  stopped 
abruptly.  On  May  27  twelve  specimens  were  taken  at  random. 
In  all  a  bacteriophage  active  for  B.  gallinarum  was  found  (in  1 
+  +  +  +  ,  in  9  +  +  +-,  in  2  ++). 

A  third  example  may  be  mentioned,  in  which  the  infection  was 
a  paratyphosis.6  On  October  15  strains  of  B.  pfaffi  were  isolated 
from  two  specimens  of  blood,  taken  from  animals  which  had  died 
in  a  chicken-yard  where  for  about  a  month  there  had  been  an 
infection  presenting  the  characters  of  typhosis.  From  specimens 
of  the  feces  taken  from  two  healthy  animals  living  in  the  same 
yard  two  strains  of  bacteriophage  were  isolated,  one  showing  a 
low  virulence  (+)  for  B.  pfaffi,  the  other  showing  no  activity  for 
this  bacillus.  Towards  the  end  of  the  month  three  chickens 
became  sick,  recovered  after  an  interval  of  two  or  three  days,  and 
then  the  epizootic  ceased.  Six  specimens  of  feces  examined  at 
this  time  all  showed  a  bacteriophage  of  high  virulence  (+  +  +) 
for  B.  pfaffi.  Against  B.  gallinarum  four  were  inactive  and  two 
showed  a  weak  virulence  (-}-). 

B.  pfaffi  was  therefore  the  cause,  for  when  the  epizootic  broke 
out  three  months  later  the  eighty  chickens  which  had  survived 
received  a  subcutaneous  injection  of  0.5  cc.  of  a  culture  of  the 
anti-pfaffi  bacteriophage  and  the  epidemic  stopped  abruptly 
and  permanently  from  the  time  of  the  injection.  We  will  see 
later  that  this  abrupt  cessation  is  the  rule  following  immuniza- 
tion by  means  of  a  culture  of  the  bacteriophage. 

6  These  experiments  were  carried  out  with  the  assistance  of  M.  Micheau, 
D.  V.  M.  at  Trainel  (Aube). 


THE   BACTERIOPHAGE   IN   DISEASE  211 

These  facts  can  be  explained  in  only  one  way.  A  weak  or 
moderate  activity  of  the  intestinal  bacteriophage  for  the  patho- 
genic bacterium  is  sufficient  to  render  the  animal  resistant  to 
infection.  The  pathogenic  bacteria  which  are  able  to  penetrate 
into  the  intestine  are  destroyed  before  they  can  multiply.  But 
it  is  not  the  same  once  the  disease  has  appeared  and  the  organism 
is  invaded.  The  animal  recovers,  and  this  is  very  rare  in  typhosis, 
only  because  of  a  rapid  adaptation  of  the  bacteriophage  and  the 
acquisition  of  a  high  virulence  which  leads  to  an  intensive  destruc- 
tion. This  bacteriophage  with  exalted  virulence  is  distributed 
broadcast  with  the  excreta  of  the  recovered  or  convalescent  ani- 
mals, and  continues,  indeed,  during  several  months  after  recovery. 
This  bacteriophage  is  necessarily  ingested  by  the  other  animals 
of  the  barn-yard  which  become,  in  fact,  "infected"  by  an  ex- 
tremely active  bacteriophage  and  by  this  means  acquire  a  complete 
protection  against  the  disease,  in  spite  of  the  presence  of  the 
pathogenic  organism  in  the  environment,  and  in  spite  of  its  fre- 
quent ingestion,  an  ingestion  which  serves  to  maintain  the  viru- 
lence of  the  bacteriophage. 

These  hypotheses  are  not  simply  idle  speculation,  for  the 
interpretation  given  to  these  observed  facts  is  confirmed  by  ex- 
periments which  provide  in  a  controlled  manner  the  natural 
conditions  of  the  epizootic.  Furthermore,  it  will  be  seen  that 
the  role  of  defense  assigned  to  the  bacteriophage  is  confirmed  by 
the  immunization  of  several  thousand  animals  by  the  adminis- 
tration of  cultures  of  an  active  bacteriophage. 

Before  discussing  these  control  experiments  I  ought  to  mention 
that,  thanks  to  the  kindness  of  the  veterinarians  of  different 
regions  invaded  by  typhosis,  I  have  been  able  to  procure  numer- 
ous specimens  of  blood  and  excreta  taken  from  sick  chickens,  from 
chickens  which  had  died  or  from  those  which  had  recovered,  de- 
rived from  eleven  different  foci  scattered  throughout  all  France. 
This  allows  me  to  generalize  from  the  facts  that  I  have  personally 
observed. 

Control  experiments 

The  control  experiments  have  been  conducted  in  Paris,  that  is 
to  say,  entirely  outside  of  the  epizootic  area. 


212  THE   BACTERIOPHAGE 

Six  chickens,  procured  from  a  region  free  of  infection,  were 
placed  under  observation.  Their  excreta  were  examined  daily 
for  ten  days  for  the  purpose  of  establishing  the  complete  absence 
of  a  bacteriophage  active  for  B.  gallinarum. 

Chicken  no.  1  then  received,  per  os,  1  cc.  of  a  culture  of  a  strain 
of  bacteriophage  very  active  for  B.  gallinarum  (+  +  ++). 

Chicken  no.  2  received  0.5  cc.  of  the  same  culture  by  subcutaneous 
injection. 

The  next  day  examination  of  the  feces  of  these  two  animals 
showed  the  presence  of  a  bacteriophage  strongly  virulent  for 
B.  gallinarum.  Therefore,  the  bacteriophage  passed  into  the 
intestine,  whether  ingested  or  injected.  This  same  fact  has  since 
been  verified  with  man  and  with  different  animals. 

Chicken  no.  1  next  received  per  os  daily  for  twenty-five  days, 
2  cc.  of  a  bouillon  culture  of  B.  gallinarum.  The  active  bac- 
teriophage persisted  in  the  intestine  with  its  primary  virulence 
(+  +  ++)  and  maintained  itself  up  to  nine  days  after  the  last 
dose  of  the  pathogenic  organism. 

Chicken  no.  2,  which  had  received  nothing  after  the  inocula- 
tion of  the  active  bacteriophage  ceased  to  show  an  active  strain 
for  B.  gallinarum  within  three  days  after  the  injection.  In  other 
words,  chicken  no.  1,  subjected  to  repeated  reinfections,  retained 
an  intestinal  bacteriophage  active  for  B.  gallinarum  for  thirty- 
four  days,  while  chicken  no.  2,  not  infected,  for  only  three  days. 

It  follows  that  the  intestinal  bacteriophage  remains  active 
only  if  it  is  able  to  develop  in  the  intestine  at  the  expense  of  this 
bacterium,  but  in  such  a  case  it  remains  active  just  so  long  as 
this  condition  is  fulfilled.  Inversely,  the  presence  in  the  intestine 
of  a  bacteriophage  possessing  virulence  for  a  given  bacterium 
indicates  that  this  bacterium  was  a  short  time  previously  in  the 
intestine. 

In  the  course  of  the  preceding  experiment  chickens  nos.  3  and 
4  were  placed  in  contact  with  chicken  no.  1.  They  all  ate  and 
drank  from  the  same  containers,  the  more  so  since  they  were 
changed  about  in  the  pens  in  such  a  manner  as  to  simulate  condi- 
tions of  life  analogous  to  those  of  the  chicken-yard.  Two  days 
after  the  first  contact,  in  the  case  of  chicken  no.  3,  three  days  after 
with  chicken  no.  4,  their  ex^r^ta  contained  a  bacteriophage  very 


THE   BACTERIOPHAGE  IN  DISEASE  213 

virulent  for  B.  gallinarum  (H — h  ++).  From  this  time  on  they 
each  received  each  day  for  twenty-one  days,  2  cc.  of  a  bouillon 
culture  of  B.  gallinarum.  At  no  time  did  they  appear  sick.  The 
intestinal  bacteriophage  remained  active  for  the  bacillus  through- 
out the  entire  period  of  the  administration  of  the  pathogenic 
bacillus,  and  even  longer — seven  days  in  no.  4  and  ten  days  in  no.  3. 
The  intestinal  bacteriophage  did  not  then  disappear,  for  as  in 
the  case  of  chickens  nos.  1  and  2,  it  remained  active  for  one  or 
several  members  of  the  colon-typhoid-dysentery  group.  But 
the  virulence  for  B.  gallinarum  did  not  persist  when  the  ingestion 
of  cultures  of  this  last  bacillus  was  stopped.  The  experiment 
with  chickens  nos.  3  and  4  shows  clearly  that  the  bacteriophagous 
ultramicrobe  is  infectious  in  exactly  the  same  sense  as  is  the 
pathogenic  bacillus  itself,  since  these  birds  were  ''contaminated" 
by  contact  with  chicken  no.  1. 

Chickens  nos.  5  and  6,  which  had  not  been  in  contact  with  the 
other  chickens,  and  which  on  repeated  examinations  were  shown 
to  be  free  of  a  bacteriophage  active  for  B.  gallinarum,  each  re- 
ceived per  os,  on  some  bread,  a  single  dose  of  2  cc.  of  a  bouillon 
culture  of  B.  gallinarum.  Three  days  after  the  infecting  meal 
diarrhea  appeared  and  they  died  two  and  three  days  later,  after 
having  shown  all  of  the  symptoms  of  the  natural  disease.  Ne- 
cropsy showed  the  presence  of  the  same  lesions.  Cultures  of 
the  blood  gave  pure  cultures  of  the  pathogenic  bacillus,  which 
was  likewise  found  in  abundance  in  the  intestinal  contents. 

Chickens  nos.  1,  3,  and  4,  which  had  resisted  repeated  inges- 
tions  of  B.  gallinarum  culture  without  showing  the  least  incon- 
venience, were  therefore  immunized;  the  first  as  a  result  of  the 
ingestion  of  a  bacteriophage  active  for  the  pathogenic  bacterium, 
the  two  others  by  simple  association  with  the  first. 

About  one  month  after  the  virulence  of  the  bacteriophage  for 
B.  gallinarum  had  disappeared  in  chickens  nos.  1,  2,  3  and  4 
each  of  them  was  given  on  each  of  three  days  2  cc.  of  a  culture  of 
the  bacillus.  In  all  the  intestinal  bacteriophage  showed  a  new 
virulence  for  the  pathogenic  organism.  None  of  them  showed 
the  slightest  trouble. 

In  all  of  these  experiments  the  infections  have  been  made  with 
bouillon  cultures  of  B.  gallinarum  prepared  directly  from  the 


214  THE   BACTERIOPHAGE 

blood  of  chickens  dead  of  spontaneous  natural  infection.  This 
is  essential  because  of  the  loss  in  virulence  of  this  organism  which 
takes  place  under  artificial  cultivation. 

With  chickens  nos.  5  and  6  the  ingestion  of  the  pathogenic 
bacillus  caused  a  fatal  attack  of  typhosis.  The  intestinal  bac- 
teriophage  at  no  time  manifested  an  activity  for  the  causative 
organism.  In  chickens  nos.  1,  2,  3,  and  4,  on  the  contrary,  the 
ingestion  of  the  same  culture  caused  no  disturbance  and  their 
intestinal  bacteriophage  which  for  about  a  month  had  showed  no 
activity  for  the  bacillus,  rapidly  recuperated  its  first  activity. 
It  had,  therefore,  not  disappeared  from  the  intestine,  although 
its  activity  was  no  longer  evident,  but  when  it  found  itself  again 
in  contact  in  the  intestine  with  the  pathogenic  organism  it  rapidly 
regained  its  potency. 

This  "  latent  virulence"  may  be  maintained  for  a  very  long 
time.  In  this  connection  I  may  recall  the  fact  cited  of  a  strain  of 
bacteriophage  still  possessing  after  three  years  and  more  than 
1000  passages  in  vitro,  always  with  the  Shiga  bacillus,  the  power 
to  attack  B.  coli  and  B.  typhosus.  It  showed  a  weak  power,  but 
was  capable  of  rapid  augmentation  by  transfers  at  the  expense 
of  these  organisms.  This  is  exactly  what  this  experiment  shows 
us  to  take  place  in  vivo  in  the  chicken. 

Can  a  chicken  contract  typhosis  in  spite  of  the  presence  of  an 
active  bacteriophage  in  the  intestine?  It  certainly  can.  As 
we  have  seen  in  many  experiments  the  bacterium  may  develop 
a  resistance  to  the  action  of  the  bacteriophage  and  this  resistance 
is  one  of  the  factors  comprising  the  virulence  of  the  bacterium. 
We  have  then,  on  the  one  hand,  the  bacterium,  which  when  in- 
troduced into  the  organism  may  acquire  a  resistance  to  the  action 
of  the  bacteriophage  ranging  from  zero  to  absolute  resistance, 
and  on  the  other  hand,  the  bacteriophage,  which  at  the  same  time 
may  possess  a  virulence  running  from  zero  to  extreme  activity. 
Infection  occurs,  or  does  not  occur,  according  to  whether  the 
algebraic  sum  of  virulence  -f  resistance  is  in  favor  of  the  one  or 
the  other  of  the  two  organisms  present.  Once  the  disease  has 
manifested  itself,  the  virulence  of  the  one  and  the  resistance  of 
the  other  become  increased  or  attenuated  according  to  the  con- 
ditions of  the  moment  and  the  aptitudes  previously  acquired 


THE   BACTERIOPHAGE   IN   DISEASE  215 

favoring  the  one  or  the  other  of  the  two  germs.     The  sequence  in 
which  the  events  of  the  struggle  unfold  determine  the  issue. 

Conclusions 

The  observations  made  in  natural  disease  and  the  experiments 
which  confirm  the  deductions  which  these  observations  suggest, 
show  that  the  bacteriophagous  ultramicrobe  is  always  present 
in  the  intestine  of  the  chicken,  whether  it  is  healthy  or  sick,  whether 
it  lives  in  a  locality  free  of  infection  or  in  an  epizootic  zone. 

Against  a  definite  bacterium,  B.  gallinarum  in  so  far  as  avian 
typhosis  is  concerned,  the  intestinal  bacteriophage  may  be  viru- 
lent or  avirulent,  and  in  the  first  case  its  virulence  may  be  ex- 
ercised according  to  a  scale  which  passes  from  the  smallest  degree 
capable  of  detection  to  one  of  extreme  activity. 

Virulence  of  the  bacteriophagous  ultramicrobe  for  B.  galli- 
narum is  only  observed  in  an  infected  locality.  The  absence  of 
such  a  virulence  is  equally  the  rule  with  animals  which  are  about 
to  die  and  with  those  which  have  died. 

In  a  contaminated  area  animals  which  harbor  in  their  intestine 
a  bacteriophage  endowed  with  sufficient  virulence  for  the  patho- 
genic bacterium  are  by  this  very  fact  protected  against  the  dis- 
ease, and  they  remain  so,  provided  the  actual  or  latent  virulence 
is  maintained  at  a  level  sufficiently  high  to  effect  a  rapid  destruc- 
tion of  the  pathogenic  bacilli  ingested. 

The  ingestion  of  pathogenic  bacilli  at  sufficiently  frequent 
intervals  constitutes  the  principal  factor  in  maintaining  the 
virulence  for  the  given  bacterium.  Among  the  factors  which 
contribute  to  diminishing  the  virulence  or  causing  the  virulence 
of  the  bacteriophage  for  a  pathogenic  bacterium  to  disappear, 
I  would  place  as  most  significant  the  introduction  into  the  or- 
ganism of  bacteria  endowed  with  resistance  to  the  action  of 
the  bacteriophage.  We  have  clearly  seen  this  fact  in  the  course 
of  the  experimental  study  of  the  .  phenomenon  of  the  resistance 
of  bacteria.  Another  possible  factor,  influencing  the  activity 
of  the  bacteriophage  is  the  reaction  of  the  medium  in  the  intestine, 
which  may  vary  according  to  the  accidental  conditions  of  the 
moment,  the  type  of  food,  etc.  The  importance  of  the  reaction 
of  the  medium  has  already  been  shown  for  lysis  in  vitro. 


216  THE   BACTERIOPHAGE 

A  bacteriophage  which  has  lost  its  virulence  for  the  pathogenic 
bacterium  lacks  the  power  to  exercise  it  because  of  the  absence 
of  this  bacterium,  but  it  possesses  nevertheless,  a  latent  viru- 
lence. When  placed  again  after  a  greater  or  less  length  of  time 
in  the  presence  of  this  bacterium  it  regains  its  original  virulence. 

The  fact  of  the  habitual  virulence  of  the  intestinal  bacterio- 
phage for  B.  gallinarum  in  the  infected  regions  indicates  the 
frequency  of  the  ingestion  of  these  bacilli,  and  consequently  the 
excessive  contamination  of  the  environment  by  the  pathogenic 
organism. 

In  contaminated  regions  the  animal  in  which  the  intestinal 
bacteriophage  does  not  enjoy  any  activity  for  B.  gallinarum, 
quickly  contracts  the  disease.  It  may  resist  and  recover,  but 
this  is  the  exception,  occurring  only  when  the  intestinal  bacterio- 
phage quickly  acquires  a  virulence  for  the  infecting  bacillus.  In 
the  contrary,  and  usual,  case  the  animal  succumbs. 

In  a  chicken  which  recovers,  the  intestinal  bacteriophage  ac- 
quires a  considerable  virulence  against  the  pathogenic  bacterium 
and  maintains  this  for  a  very  long  time;  in  fact,  as  long  as  the 
exterior  environment  remains  infected.  This  persistence  of  viru- 
lence is  maintained  by  the  frequent  ingestion  of  pathogenic  or- 
ganisms, which  allow  the  bacteriophage  to  multiply  at  the  expense 
of  the  particular  organism.  The  resistant  animal  disseminates 
in  its  excreta  the  bacteriophage  of  enhanced  virulence;  the  ani- 
mals which  associate  with  it  become  "  contaminated"  and  by  this 
fact  they  enter  the  same  class  of  resistant  animals  as  those  which 
have  recovered.  Recovery  of  one  animal  in  a  barn-yard  often 
marks  the  end  of  an  epizootic,  or  its  arrest  for  a  few  months. 

The  study  of  an  epidemic  of  avian  typhosis  shows,  in  a  word, 
that  the  history  of  the  contagion  reflects,  in  the  last  analysis,  the 
story  of  the  struggle  between  the  two  agents — the  pathogenic 
bacterium  and  the  bacteriophagous  ultramicrobe — and  since 
this  last  is  transmissible  from  individual  to  individual  the  immunity 
is  contagious  in  the  same  sense  as  the  disease  itself.  The  be- 
ginning of  an  epizootic  is  marked  by  a  diffusion  of  the  bacteria, 
the  end  by  a  diffusion  of  a  bacteriophage  virulent  for  these  bac- 
teria. We  will  encounter  the  same  facts  in  another  disease;  in 
hemorrhagic  septicemia  in  the  buffalo. 


THE   BACTERIOPHAGE   IN  DISEASE  217 

HEMORRHAGIC  SEPTICEMIA  OF  THE  BUFFALO  (BARBONE)7 

Barbone,  the  disease 

Contrary  to  the  diseases  discussed  up  to  this  point,  barbone 
does  not  present  intestinal  symptoms;  it  is  of  the  hemorrhagic 
septicemia  type.  The  pathogenic  organism  is  a  Pasteurella. 
Cultures  of  the  organism  in  beef  bouillon  maintain  their  virulence 
for  a  considerable  time — at  least  eighteen  months.  The  inocula- 
tion of  a  buffalo  or  of  a  cow  with  0.0002  cc.  of  a  virulent  culture 
kills  the  animal  in  between  thirty-six  and  forty  hours  with  all 
the  symptoms  of  the  spontaneously  acquired  disease.  At  ne- 
cropsy identical  lesions  are  found  and  the  pathogenic  bacterium 
swarms  in  the  blood  and  in  the  organs.8 

The  buffalo  is  par  excellence  the  beast  of  burden  in  the  culti- 
vation of  rice-fields;  it  replaces  the  ox  in  all  southern  Asia  and 
in  the  islands  of  the  Sunda  Straits.  It  is  utilized  in  certain 
regions  of  Italy,  in  Egypt,  in  Hungary,  and  in  the  Balkans.  Wher- 
ever the  buffalo  lives  there  also  will  be  found  barbone,  the  most 
terrible,  without  doubt,  of  all  the  contagious  diseases.  The  re- 
ports indicate  a  mortality  of  from  70  to  95  per  cent.  I  was 
present  during  an  epizootic  which  raged  in  June,  1920,  in  the 
Province  of  Bac  Lieu  (Cochin-China)  where  among  the  thirty 
thousand  buffaloes  of  the  region  ten  thousand  died,  and  I  did  not 
have  an  opportunity  to  observe  a  single  animal  which  recovered. 
Recovery  may  occur,  but  it  is  certainly  rare,  and  the  mortality 
in  Cochin-China  is  certainly  above  99  per  cent  of  the  animals 
affected. 

The  average  duration  of  the  evolution  of  the  disease  is  but 
eighteen  to  twenty-four  hours;  rarely  thirty-six.  Death  some- 
times takes  place  without  precursory  symptoms.  An  animal  yoked 
to  a  plow  stops,  remains  motionless  for  a  few  moments  with  an 

7  The  experiments  on  barbone  have  been  performed  in  collaboration 
with  G.  Le  Louet,  Chief  of  the  Veterinary  Service  in  Cochin-China. 

8  In  two  different  attempts  I  have  proved  that  diluted  blood  or  macera- 
tions of  organs  (liver  and  lung)  taken  from  animals  dead  of  spontaneous 
infection,  filtered  through  a  Chamberland  filter  (L2)  and  inoculated  in 
large  amounts  into  the  buffalo  or  into  cattle  do  not  cause  the  slightest  dis- 
ease symptoms. 


218  THE   BACTERIOPHAGE 

haggard  aspect  and  then  falls  as  though  struck  by  lightning.  In 
typical  cases,  which  can  be  reproduced  in  a  perfect  manner  in 
experimental  infection,  the  animal  appears  dejected,  the  eyes 
fixed,  the  head  lowered.  The  temperature  rapidly  mounts  to 
41.5  to  42.5°C.,  the  respiration,  at  first  accelerated,  becomes 
slowed  and  then  dyspneic,  the  inspirations  less  and  less  frequent. 
The  animal  shows  meteorism;  it  lies  flat  on  the  ground  in  complete 
lateral  decubitus  usually  a  short  time  before  death  which  is  pre- 
ceded by  cramps  and  at  times  convulsions. 

Often  tumefaction  is  to  be  observed,  appearing  usually  in  the 
region  of  the  throat  and  extending  back  to  the  shoulder.  The 
engorgement  is  produced  by  a  gelatinous  exudate  of  a  yellow 
color  within  the  connective  tissue.  At  times  the  tumefaction 
appears  in  another  part  of  the  body,  or  it  may  be  entirely  lacking. 
This  tumefaction,  as  shown  in  experimental  infection,  marks  the 
portal  of  entrance  of  the  pathogenic  bacteria.  Infection  usually 
occurs  by  way  of  the  digestive  tract  and  the  virus  most  frequently 
penetrates  the  tissues  through  some  portion  of  the  nasopharynx. 
A  tumefaction  on  another  part  of  the  body — thigh,  abdomen, 
rump — indicates  a  reinfection  by  the  penetration  of  the  virus 
through  an  excoriation.  Examination  of  cadavers  shows  that 
the  absence  of  tumefaction  indicates  an  infection  by  way  of  the 
stomach  and  intestine. 

Bovines  and  the  buffalo  are  equally  susceptible,  as  was  noted 
a  long  time  ago  by  Piot  in  Egypt.  The  statistics  of  Indo-China 
indicate,  it  is  true,  that  the  mortality  from  barbone  is  but  slight 
for  cattle,  but  this  is  solely  due  to  the  fact  that  these  animals  are 
present  in  but  small  numbers  in  the  regions  where  barbone  rages; 
regions  which  are  extremely  humid  and  admirably  adapted  to 
the  buffalo,  a  semi-aquatic  animal.  The  rare  cattle  found  some- 
times in  such  regions  contract  the  disease  and  die  like  the  buffalo, 
after  having  presented  identical  symptoms. 

The  effect  of  low  places  and  swamps  on  the  contagion  has  been 
from  time  immemorial  recognized  by  the  natives.  When  it  is 
possible,  as  soon  as  a  case  of  barbone  is  detected  in  a  neighborhood, 
they  hasten  to  collect  their  animals  and  remove  them  to  a  more 
elevated  region.  It  is  known,  moreover,  that  the  organisms  of 
the  Pasteurella  group  remain  virulent  for  a  very  long  time  in 
the  mud  of  the  marshes  and  in  the  slime  of  the  streams. 


THE   BACTERIOPHAGE   IN   DISEASE  219 

Rdle  of  the  bacteriophage  in  the  disease 

In  Cochin-China  barbone  is  always  present  in  sporadic  form 
causing  each  year  numerous  small  epizootics  which  remain  lo- 
calized in  individual  villages.  A  localized  epidemic  observed 
in  Long  Huu  in  the  Province  of  Gocong  may  serve  as  an  example. 

From  May  5  to  13,  1920,  seventeen  buffaloes  died: — on  May  5, 
one;  May  7,  three;  May  8,  two;  May  9,  one;  May  10,  two;  May 
11,  four;  May  12,  three;  and  May  13,  one.  Then  the  epizootic 
stopped  and  not  a  single  case  was  detected  during  the  next  six 
months. 

Specimens  of  the  feces  of  four  of  these  animals  were  collected, 
either  before  death  or  from  the  cadaver.  None  contained  a  bac- 
teriophage  active  for  the  bacterium  of  barbone.  On  May  13 
specimens  of  feces  were  collected  from  healthy  animals,  as  follows : 

First.  From  a  buffalo  in  a  stable  where  two  animals  had 
died,  one  on  May  12,  the  other  on  May  13. 

Second.  From  three  buffaloes  in  a  stable  where  one  had  died 
on  May  5. 

Third.  From  two  buffaloes  in  a  stable  where  two  had  died, 
one  on  May  8,  the  other  on  May  11. 

Fourth.  From  four  buffaloes  in  a  stable  which  had  not  been 
invaded. 

Fifth.  From  one  buffalo,  living  alone  in  a  stable  located  at 
a  distance  of  about  five  kilometers  from  the  village  of  Long  Huu. 

Sixth.  From  eight  buffaloes  in  the  surrounding  villages,  from 
eleven  to  nineteen  kilometers  distant. 

Of  all  the  specimens,  those  in  the  first,  second,  third  and  fourth 
groups  gave  a  bacteriophage  of  weak  or  average  activity  (+  or 
++)  for  the  bacterium  of  barbone.  An  active  bacteriophage 
was  not  found  in  the  specimens  from  groups  5  and  6. 

Again  on  May  19  specimens  were  collected  in  Long  Huu,  as 
follows: 

First.  From  the  buffalo  which  had  furnished  specimen  no.  1 
on  May  13. 

Second.  From  two  buffaloes  living  in  a  stable  where  three 
had  died  from  May  7  to  May  12.  These  specimens  all  gave  a 
bacteriophage  moderately  virulent  (++)  for  the  bacterium  of 
barbone. 


220  THE   BACTERIOPHAGE 

The  animals  which  resisted,  therefore,  showed  in  their  intestine 
a  bacteriophage  virulent  for  the  pathogenic  bacterium. 

The  epizootic  does  not  always  remain  localized  in  a  village. 
At  times  it  spreads  rapidly  from  village  to  village  and  within  a 
few  days  will  extend  over  a  very  considerable  territory.  It  is 
rarely  possible  to  determine  the  primary  focus,  so  great  is  the 
speed  with  which  it  spreads.  The  mortality  then  becomes  con- 
siderable, the  losses  often  amount  to  tens  of  thousands  of  animals, 
as  has  been  observed  many  times  in  China,  in  British  India,  and 
in  the  Dutch  East  Indies.  Sometimes  even,  as  actually  happened 
in  Java,  the  buffalo,  as  a  race,  is  practically  eliminated. 

In  the  first  two  weeks  of  June  1920  the  epizootic  became  general 
in  the  Province  of  Bac  Lieu  and  in  certain  parts  of  the  adjacent 
provinces  (western  Cochin-China).  It  was  possible  to  examine 
the  blood  of  eleven  animals  which  died  in  widely  scattered  parts 
of  the  area  invaded,  and  in  all  the  bacterium  of  barbone  was  found 
in  considerable  quantity.9  The  epizootic  died  out  during  the  first 
fortnight  of  July.  It  had  persisted  for  a  month,  killing  a  third 
of  the  animals  in  the  district. 

The  region  of  Thoi  Binh  was  particularly  affected,  the  loss 
amounting  to  more  than  fifty  per  cent  of  the  buffaloes  in  the 
locality.  From  July  8  to  13,  at  the  time  when  the  epizootic  was 
disappearing  (the  last  animal  to  be  affected  died  on  July  12), 
twenty  specimens  of  feces  were  collected.  These  were  taken  from 
buffaloes  which  had  resisted  the  infection  and  which  at  no  time 
showed  any  evidence  of  the  typical  symptoms  of  the  disease. 
All  of  the  animals  examined  lived  on  the  farms  of  the  village 
of  Thoi  Binh  or  in  the  neighboring  hamlets  within  a  radius  of 
fifteen  kilometers. 

Tests  for  the  virulence  of  the  intestinal  bacteriophage  against 
the  bacterium  of  barbone  gave  the  following  results: 

9  Bacteriological  diagnosis  is  easy,  even  if  the  only  available  material 
is  some  blood  or  a  fragment  of  an  organ  taken  without  any  special  pre- 
cautions in  the  field,  as  is  usual  in  such  countries.  Even  if  the  specimen 
is  some  days  old  it  is  only  necessary  to  smear  it  over  the  shaved  skin  of  a 
rabbit.  If  the  bacterium  of  barbone  is  present  the  animal  will  die  within 
24  hours,  and  the  organism  will  be  found  in  pure  culture  in  the  blood,  from 
which  it  may  be  readily  isolated.  This  is  also  the  best  method  for  detecting 
the  bacterium  in  soil  or  in  fecal  material. 


THE   BACTERIOPHAGE   IN   DISEASE 


221 


FARM 

MORTALITY 

THE  LAST 
ANIMAL  DIED 
ON 

NUMBER 
OF  A.NIMAL8 
WHICH 
RESISTED 

VIRULENCE 
OF  THE 
BACTERIO- 
PHAGE 

Ncau  1              

3 

July  7 

10 

+  + 

Ngau  2  

+ 

Ngau  3      

+4--h 

De  

2 

'    June  10 

1 

-}- 

Doi  1  

5 

June  28 

4 

++++ 

Doi  2           

++  + 

La/nli                  

9 

July  2 

4 

-f-  \- 

Tran  

1 

July  4 

2 

++++ 

The            

3 

July  11 

1 

+++-f 

H  v  Chanh        

2 

July  2 

2 

H-+++ 

Hien    

0 

4 

++4  + 

Du                   

0 

6 

+ 

Sam  

2 

July  2 

8 

++ 

P  v  Chanh  

6 

June  30 

8 

+-f  +  + 

Cu                          

1 

July  2 

5 

++-f 

So  

3 

July  6 

5 

++ 

No                   

5 

June  30 

10 

++  + 

Phuc 

8 

July  3 

4 

+  ++4- 

Gia      

8 

June  29 

3 

+++ 

Man                      

1 

June  30 

3 

++ 

From  this  it  appears  that  the  intestinal  bacteriophage  is  en- 
dowed with  virulence  for  the  bacterium  of  barbone  in  all  the 
buffaloes  which  the  disease  had  spared. 

In  the  course  of  different  trips  across  Indo-China,  I  collected 
forty-one  specimens  of  feces  from  buffaloes,  each  specimen  col- 
lected in  a  different  village  in  which  no  buffaloes  had  died  of 
barbone  for  at  least  two  years.  In  only  three  of  these  specimens 
could  a  bacteriophage  active  for  the  bacterium  of  barbone  be 
demonstrated,  and  in  these  cases  it  was  weak  (+)•  Nevertheless, 
the  intestinal  bacteriophage  was  present  in  all;  but  although  it 
was  active  for  one  or  another  of  the  intestinal  organisms,  its  viru- 
lence was  weak  or  lacking  for  the  bacterium  of  barbone. 

We  will  see  later,  on  the  contrary,  that  in  a  contaminated  area 
at  the  time  when  the  epizootic  dies  out,  the  intestinal  bacterio- 
phage of  all  of  the  buffaloes  which  escaped  the  disease  is  virulent 
for  the  bacterium,  the  causative  agent  of  the  epizootic.  We  find 
here,  then,  the  same  facts  as  in  the  previous  disease  studied; 


222  THE   BACTERIOPHAGE 

that  the  protection  of  the  body  in  the  case  of  barbone,  a  septicemic 
disease,  is  assured  by  the  bacteriophage. 

In  the  buffaloes  of  a  region  ravaged  by  the  disease  the  bacterio- 
phage preserves  for  a  very  long  time  its  virulence  for  the  patho- 
genic bacterium.  This,  the  following  example  shows. 

In  November,  1919,  a  localized  epizootic  of  barbone  occurred 
among  the  buffaloes  of  the  village  of  Phuoc  Thien  (Province  of 
Bien  Hoa).  On  a  farm  having  twenty-one  buffaloes  seven  died 
— two  adult  animals  and  five  aged  from  one  to  two  years.  The 
disease  died  out,  or  to  speak  more  correctly,  after  this,  two  ani- 
mals recovered  one  after  another.  On  the  12th  of  the  following 
April,  that  is  to  say,  five  months  later,  specimens  of  the  feces  of 
eight  of  the  surviving  animals  were  collected.  All  contained  a 
bacteriophage  active  for  the  bacterium  of  barbone  (six  were  +  +  > 
two  were  +)• 

Two  specimens  of  the  mud  of  a  water-hole  where  the  animals 
were  accustomed  to  remain  immersed  up  to  the  neck  during  the 
hottest  hours  of  the  day  were  also  examined.  In  both  a  bacterio- 
phage virulent  (++)  for  the  bacterium  of  barbone  was  found. 
The  destruction  of  the  pathogenic  bacterium  in  the  external 
medium  must  often  be  effected  by  the  bacteriophage,  for  it  is 
certain  that  if  the  bacterium  of  barbone  has  once  been  introduced 
into  a  water-hole  by  a  sick  animal  the  bacteriophage  present 
there  must  destroy  it.  Furthermore,  this  fact  shows  one  of  the 
modes  of  "  contagion"  of  the  active  bacteriophage.  A  single 
buffalo,  in  the  intestine  of  which  the  bacteriophage  has  acquired 
a  virulence  for  the  pathogenic  bacterium,  is  sufficient  to  "con- 
taminate" all  the  herd  which  frequent  the  water-hole.  Localized 
epizootics  are  of  short  duration,  but  in  spite  of  this  we  find  that 
the  pathogenic  bacterium  persists  for  several  months  in  the  ex- 
ternal world  and  that  their  ingestion  by  buffaloes  is  frequent, 
since  the  virulence  of  the  bacteriophage  maintains  itself  against 
this  bacterium.  The  repeated  ingestion  of  a  bacterium  is,  as 
we  have  seen,  essential  for  the  permanence  of  the  virulence  of 
the  bacteriophage  toward  this  bacterium.  The  epizootic  dies 
out,  not  because  of  an  absence  of  pathogenic  bacteria  but  because 
of  the  presence  of  a  virulent  bacteriophage  in  the  intestine  of 
all  exposed  animals. 


THE   BACTERIOPHAGE   IN   DISEASE  223 

All  of  the  observations  are  therefore  comparable,  whether 
they  deal  with  avian  typhosis  or  with  barbone  in  the  buffalo. 
These  epizootics  of  very  different  nature  were  investigated  in- 
tentionally, that  the  general  nature  of  the  role  of  the  bacterio- 
phage  in  immunity  might  be  the  better  established. 

One  may  at  first  be  quite  astonished  that  the  intestinal  bac- 
teriophage,  whose  role  can  easily  be  conceived  in  infections  with 
intestinal  manifestations,  constitutes  a  defense  of  the  organism 
in  septicemias.  In  reality,  whatever  may  be  the  infection,  the 
pathogenic  bacterium  always  gets  into  the  intestine.  Let  us 
take  a  localized  disease,  cerebrospinal  meningitis,  for  example. 
We  know  that  the  initial  symptom  is  a  rhino-pharyngitis  and 
that  even  healthy  subjects  who  have  been  in  contact  with  a 
patient  often  carry  the  specific  germ  in  the  nasopharynx.  There 
can  be  no  doubt  but  that  a  fair  number  of  the  meningococci 
present  in  the  rhino-pharynx  are  swallowed  and  pass  into  the 
intestine.  It  is  needless  to  insist  on  this,  that,  aside  from  a  few 
rare  exceptions  to  which  we  will  later  return,  whatever  may  be 
the  disease  under  consideration,  the  portal  of  entrance  of  the  virus 
is  either  the  buccal  route  or  by  way  of  the  respiratory  tract. 
In  either  case  the  ingestion  of  organisms  is,  it  might  be  said,  ob- 
ligatory. The  pathogenic  bacterium  is  always  at  some  time  in 
contact  with  the  intestinal  bacteriophage,  this  organism  there- 
fore is  thus  able  to  adapt  itself  to  the  bacteriophagy  of  the  bac- 
terium and  to  acquire  a  virulence. 

In  the  particular  case  of  barbone  the  pathogenic  bacterium  is 
found  freely  disseminated  through  the  exterior  world  in  con- 
taminated regions.  In  an  epizootic  zone  I  have  been  able,  in 
two  different  trials,  to  isolate  it  from  the  mud  of  a  marsh  where 
the  buffaloes  were  accustomed  to  bury  themselves.  This  is  but 
natural  since  the  bacterium  of  barbone  is  found  in  the  intestinal 
tract  of  sick  animals  or  of  those  which  have  succumbed.  The 
ingestion  of  the  pathogenic  bacterium  by  the  animals  which  re- 
main immersed  for  whole  hours  in  a  mire  containing  these  or- 
ganisms is  necessarily  frequent.  If  the  animal  which  ingests 
them  has  an  erosion  at  any  point  in  the  digestive  tract  it  is  sus- 
ceptible to  infection.  Otherwise  the  bacteria  reach  the  intestine 
and  come  within  the  range  of  the  intestinal  bacteriophage  which 


224  THE  BACTERIOPHAGE 

can  then  acquire  a  virulence  for  the  virus.  If  this  takes  place  the 
animal  is  thenceforth  protected  from  the  infection  and  becomes 
a  carrier  of  the  virulent  bacteriophage.  A  diseased  animal  prop- 
agates his  disease;  an  animal  in  a  resistant  condition  propagates 
his  immunity. 

BUBONIC    PLAGUE 

Through  a  lack  of  favorable  circumstances  it  has  not  been 
possible  to  follow  the  evolution  of  the  intestinal  bacteriophage  in 
man  affected  with  plague.  The  few  cases  that  have  been  ex- 
amined have  all  been  fatal,  and  at  no  time  could  the  intestinal 
bacteriophage  be  shown  to  have  the  least  virulence  for  B.  pestis. 
The  activity  in  these  cases  remained  restricted  to  B.  coli.  How- 
ever, the  stools  of  two  convalescent  individuals  have  been  se- 
cured and  examined.  According  to  the  physicians  treating  the 
cases  the  material  was  collected  on  the  sixth  and  the  eleventh 
days  after  the  beginning  of  convalescence.  Examination  showed, 
in  the  first  case,  a  bacteriophage  of  average  virulence  (++), 
and  in  the  second  case,  one  of  feeble  virulence  (+)  for  B.  pestis. 
The  virulence  of  the  first  of  these  strains  has  been  enhanced 
in  vitro  and  the  bacteriophage  has  been  maintaioed  in  culture. 

An  attempt  was  made  to  find  a  bacteriophage  active  against 
this  bacillus  in  the  feces  of  twenty-two  natives  living  in  regions 
free  of  plague,  but  in  no  case  could  a  strain  be  isolated.  However, 
in  view  of  the  particular  mode  of  infection  in  bubonic  plague  the 
study  of  its  propagation  in  man  offers  only  a  matter  of  secondary 
interest,  at  least  from  the  epidemiological  point  of  view.  We 
know  that  an  epidemic  of  plague  in  man  is  only  consequent  to 
an  epizootic  among  rats.  That  which  it  is  interesting  to  study 
is,  therefore,  the  epizootic,  the  primary  cause  of  the  epidemic. 
In  order  to  attain  a  correct  interpretation  of  results  it  is  essential 
to  follow  the  natural  order  of  things.  From  the  point  of  view 
of  man  the  epidemic  is  obviously  the  important  fact;  from  the 
point  of  view  of  nature  this  is  but  a  secondary  incident,  for  if  we 
were  able  to  suppress  the  epizootic  the  epidemic  would  cease 
spontaneously. 

From  what  we  actually  know  about  the  epidemiology  of  plague, 
it  results  that  all  of  the  rats  living  in  a  city  where  there  has  been 


THE  BACTEBIOPHAGE  IN  DISEASE  225 

a  case  of  plague  in  man  are  the  animals  which  have  resisted  the 
contagion,  either  because  they  were  infected  and  recovered,  or 
because  they  remained  unaffected.  I  have  then,  investigated  the 
virulence  of  the  intestinal  bacteriophage  of  the  rat  toward  B. 


First.  Twenty-one  specimens  of  the  excrement  of  rats  taken 
from  towns  in  Indo-China  free  of  plague  were  examined.  The 
intestinal  bacteriophage  was  found,  active  against  one  or  another 
of  the  intestinal  bacteria,  but  it  never  showed  any  virulence  what- 
ever for  B.  pestis. 

Second.  A  small  epidemic  of  plague  (eleven  fatal  cases)  occurred 
in  the  village  of  Bac  Lieu,  in  the  eastern  part  of  Indo-China, 
during  July,  1920.  On  the  following  6th  of  November  I  procured 
in  this  town  four  specimens  of  the  excreta  of  rats,  each  speci- 
men composed  of  some  dozens  of  particles,  and  certainly  derived 
from  several  individuals.  The  tests  for  virulence  against  B.  pestis 
gave  the  following  results: 


Specimen  derived  from  a  granary  ...................... 

Specimen  derived  from  the  embarkment  quay  ..........  ++  + 

Specimen  derived  from  a  decorticating  mill  ............  +++ 

Specimen  procured  in  the  house  of  a  native  ............  ++++ 

Those  rats  which  have  survived  an  epizootic,  therefore,  har- 
bor in  their  intestine  a  bacteriophage  possessed  of  a  high  viru- 
lence for  B.  pestis. 

Plague  has  existed  in  the  form  of  sporadic  cases  in  the  region 
of  Phantiet,  in  southern  Annam,  for  about  twenty  years.  I 
obtained  specimens  of  the  excrement  of  rats  in  the  infected  vil- 
lages, each  specimen  being  composed  of  the  feces  derived  from 
several  animals.  The  results  of  the  tests  for  the  virulence  of 
the  intestinal  bacteriophage  in  these  specimens  were: 

Village  Virulence 

Thien  Due  ..............................................  ++ 

Hung  Long  .............................................  + 

Due  Hang  ..............................................  +  +  + 

Due  Thang  .............................................  ++ 

Tri  Long  ................................................  +  4- 

PhuTay  ................................................  +  +  + 

Cu  Long  ................................................  + 


226  THE   BACTERIOPHAGE 

The  results  are  thus  identical  with  those  secured  at  Bac  Lieu, 
although  the  virulence  seems  to  be  somewhat  lower,  but  this 
can  only  be  of  relative  importance  since  in  the  present  case  each 
specimen  was  composed  of  the  excreta  taken  from  several  rats; 
the  results  then,  indicate  only  an  average. 

Is  the  bacteriophage  present  in  all  of  the  rats  of  an  infected 
region  or  only  in  a  certain  number?  At  Phantiet  I  collected  the 
excrement  of  six  young  rats,  according  to  their  weight,  aged  from 
three  to  four  weeks.  Examination  showed  that  four  of  the  speci- 
mens contained  a  bacteriophage  active  for  B.  pestis  (+)  while 
two  did  not.  These  last  two  animals  were  therefore  susceptible 
to  plague. 

From  the  results  given  above  one  may  conclude  that,  as  for 
avian  typhosis  and  for  barbone,  the  cause  of  the  resistance  against 
B.  pestis  is  the  presence  in  the  organism  of  a  bacteriophage  pos- 
sessing a  virulence  for  this  bacillus. 

How  is  the  adaptation  effected  in  the  case  of  B.  pestis?  At 
different  times  it  has  been  noted  that  the  bacillus  has  been  found 
in  the  intestinal  contents  of  victims  of  plague.  Thus,  it  is  pos- 
sible for  them  to  be  disseminated  by  the  feces  throughout 
the  external  world  where  they  may  again  be  ingested.  The 
bodies  of  dead  rats  constitute  another  mode  of  dissemination. 
These  bodies  are  often  devoured  by  the  surviving  rats  and  this 
extends  the  infection.  In  those  animals  which  resist  and  which 
are  infected  the  intestinal  bacteriophage  is  maintained  virulent 
for  the  pathogenic  bacillus.  But  observation  and  direct  experi- 
mentation have  shown  us  that  a  bacteriophage  is  only  possessed 
of  a  virulence  for  a  bacterium  when  the  ingestions  of  this  bac- 
terium are  frequent.  The  permanence  of  the  virulence  of  the 
intestinal  bacteriophage  of  the  rat  against  the  plague  bacillus 
indicates  the  persistence  of  this  bacillus  in  the  external  world, 
at  least  for  several  months  after  the  last  human  case  has  taken 
place.  Moreover,  the  -revival  of  the  epidemic  each  year  in  cer- 
tain localities,  Bac  Lieu  for  example,  shows  that  it  can  not  be 
otherwise.10 

10  Demonstration  of  the  presence  of  a  bacteriophage  active  for  B.  pestis 
in  the  rats  of  a  locality  would  in  certain  cases  be  very  useful  for  it  would 
indicate  the  presence  of  the  bacillus  in  the  exterior  world  and  the  possi- 


THE   BACTERIOPHAGE   IN   DISEASE  227 

FLACHERIE 

A  few  experiments  have  been  made  on  this  disease,  but  only 
for  the  purpose  of  determining  if  defense  against  infection  in 
invertebrates  is  also  assured  by  the  bacteriophage. 

In  a  breeding-place  in  Cochin-China  a  certain  number  of  silk 
worms  died  of  a  disease  presenting  all  of  the  characteristics  of 
flacherie.  Examination  of  the  excreta  of  the  sick  worms,  as 
well  as  of  the  cadavers,  showed  the  presence  of  a  cocco-bacillus, 
Gram-negative,  which  was  not  present  in  the  dejections  of  healthy 
worms.  The  ingestion,  on  mulberry  leaves,  of  some  of  the 
culture  of  this  cocco-bacillus  reproduced  the  disease;  eleven 
out  of  twelve  worms  dying  in  from  six  to  eleven  days  after  the 
infecting  feeding. 

Three  nitrates  were  prepared  from  the  excreta  of  healthy  worms 
living  in  the  baskets  where  the  affected  worms  were  found.  These 
three  filtrates  contained  a  bacteriophage  of  moderate  or  high 
virulence  (++,  +  ++,  +  +  +)  for  the  cocco-bacillus.  On  the 
other  hand,  two  filtrates  were  prepared,  the  one  with  the  intestinal 
contents  of  a  sick  worm,  the  other  with  the  intestinal  contents 
of  a  worm  which  had  died  of  the  infection.  Neither  contained 
a  bacteriophage  active  for  the  coccobacillus. 

These  experiments  have  not  been  carried  further,  since  the 
desired  end  had  been  attained.  They  were  adequate  to  show 
that  the  facts  observed  in  infectious  disease  in  mammals  were 
reproduced  in  an  infectious  disease  of  an  invertebrate.  From 
this  it  seems  logical  to  conclude  that  the  defense  of  the  organism 
by  the  bacteriophage  must  constitute  a  general  fact  throughout 
all  animals. 

bility  of  a  renewal  of  the  epidemic.  Such  a  demonstration  might  also  be 
useful  in  establishing  a  retrospective  or  doubtful  diagnosis.  Suppose  a  few 
suspicious  deaths  have  occurred  in  a  group  some  time  previously.  The  pres- 
ence in  the  rats  of  the  neighborhood  of  a  bacteriophage  showing  a  virulence 
for  B.  pestis  would  eliminate  all  doubt;  the  deaths  were  due  to  plague.  Or, 
the  question  of  the  nature  of  a  epizootic  among  the  rats  may  be  in  question. 
Was  the  mortality  due  to  plague?  The  demonstration  of  a  bacteriophage 
active  for  B.  pestis  either  in  the  dead  rats  or  in  those  that  have  survived 
provides  the  answer. 


228  THE   BACTERIOPHAGE 

CONCLUSIONS 

We  may  limit  ourselves  for  the  moment  to  the  following  points: 

Whatever  may  be  the  disease  considered,  the  picture  remains 
the  same;  when  a  pathogenic  bacterium  is  introduced  into  an 
organism,  one  of  two  situations  develops: 

First.  The  intestinal  bacteriophage  shows  an  activity  for 
this  bacterium,  the  latter  is  destroyed  before  it  can  develop,  and 
disease  does  not  appear. 

Second.  The  intestinal  bacteriophage  is  inactive,  the  bac- 
terium develops,  and  disease  results. 

In  the  course  of  the  disease  one  of  two  things  may  happen: 

First,  the  bacteriophage  in  contact  with  the  pathogenic  bac- 
terium may  acquire  a  virulence,  or 

Second.  The  bacterium  may  acquire  a  virulence,  in  other 
words,  may  become  resistant  to  the  action  of  the  bacteriophage. 

The  vicissitudes  in  the  struggle  between  these  two  factors  are 
reflected  in  the  condition  of  the  infected  individual.  Convales- 
cence begins  at  the  moment  when  the  virulence  of  the  bacterio- 
phage is  sufficient  to  give  it,  definitely,  the  upper  hand.  The 
disease  has  a  fatal  outcome  if  the  bacteriophage  is  inactive  as  a 
result  of  unfavorable  conditions,  or  if  the  bacterium  is  able  to 
acquire  a  refractory  state.  This  last  situation  appears  to  be 
very  infrequent,  at  least,  in  the  diseases  studied. 

In  epidemics  we  find  a  large  scale  reproduction  in  a  community 
of  individuals  of  the  struggle  which  takes  place  in  a  single  indi- 
vidual between  the  ultramicrobe  and  the  bacterium. 

The  bacteriophagous  ultramicrobe  is  transmissible  from  one 
individual  to  another  just  as  is  the  bacterium  itself.  The  his- 
tory of  an  epidemic  is,  in  the  last  analysis,  the  story  of  an  infec- 
tion with  two  microorganisms.  The  epidemic  ceases  at  the 
moment  when  all  susceptible  individuals  harbor  a  bacteriophage 
active  for  the  causative  organism  of  the  epidemic.  Either  the 
bacteriophage  has  acquired  virulence  in  the  body  of  the  indi- 
vidual who  harbors  it,  or  this  individual  has  been  "  contaminated " 
by  a  bacteriophage  which  has  acquired  a  virulence  in  another 
individual  for  the  specific  bacterium  involved. 


CHAPTER  II 
THE  BACTEBIOPHAGE  IN  THE  HEALTHY  INDIVIDUAL 

The  Bacteriophage  in  Man.  The  Bacteriophage  in  the  Horse.  The 
Bacteriophage  in  Fowls.  The  Bacteriophage  in  Diverse  Animals. 
Conclusions. 

The  experiments  conducted  on  patients  and  on  healthy  in- 
dividuals exposed  to  infection  have  shown  that  a  resistance  to 
the  infectious  agent  accompanies  the  presence  in  the  intestine 
of  an  ultramicrobial  bacteriophage  possessing  a  virulence  for 
the  causative  bacterium.  On  the  other  hand,  as  I  have  shown  in 
Part  I  of  this  monograph,  there  is  but  a  single  species  of  bacterio- 
phage, capable,  by  adaptation,  of  acquiring  virulence  for  the 
diverse  bacteria  which  it  attacks.1 

These  facts  being  true,  a  question  quite  naturally  arises.  Does 
this  bacteriophage  which  acquires  virulence  for  diverse  patho- 
genic bacteria  make  its  appearance  only  at  the  exact  moment 
when  it  is  needed?  Or  is  it,  indeed,  a  normal  inhabitant  of  the 
intestinal  canal?  Examination  of  the  feces  of  numerous  individ- 
uals belonging  to  very  varied  species  permits  an  answer  to  this 
question. 

THE  BACTERIOPHAGE  IN  HEALTHY  MEN 

Variations  in  the  virulence  of  the  intestinal  bacteriophage  in 
healthy  men  have  been  followed.  To  this  end,  in  a  first  series 
of  experiments,  specimens  of  the  stool  were  collected  every  fifteen 
days.  The  study  has  been  limited  to  testing  the  activity  of  the 
ultramicrobe  against  the  following  bacterial  species:  B.  coli, 
B.  dysenteriae  Shiga,  B.  dysenteriae  Flexner,  B.  dysenteriae  Hiss, 
B.  typhosus,  B.  paratyphosus  A,  and  B.  paratyphosus  B.  Later 

1  The  possibility  of  a  germ  being  virulent  for  a  great  number  of  different 
beings  is  not  an  exception  peculiar  to  the  bacteriophage.  It  is  only  neces- 
sary to  mention  B.  tuberculosis,  to  which  but  few  animals  are  insusceptible. 

229 


230  THE  BACTERIOPHAGE 

where  indicated,  the  tests  were  extended  to  other  bacterial 
species  of  particular  interest. 

With  the  first  examinations  a  weak  activity  (+),  especially 
for  B.  coli,  was  not  detected  or  remained  doubtful,  but  when  the 
tests  were  repeated  after  several  months,  using  a  more  satisfac- 
tory technic,  the  bacteriophage  was  clearly  demonstrated.  As 
will  be  seen  upon  examining  the  table  where  the  results  are  re- 
corded (table  1),  some  of  the  examinations  remained  negative; 
the  bacteriophage  appeared  to  be  absent.  Would  it  have  been 
the  same  if  it  had  been  possible  to  test  the  filtrate  against  all 
of  the  bacteria  which  may  be  found  in  the  intestine?  An  answer 
to  this  question  was  sought.  A  specimen  taken  on  July  1st  was 
inactive  toward  the  eight  species  of  bacteria  routinely  employed, 
and  it  was  tested  against  a  different  series  of  bacteria,  selected 
at  random.  The  filtrate  showed  a  high  activity  for  an  organism 
of  the  Salmonella  (hog  cholera)  group.  When  the  same  experi- 
mental tests  were  repeated  on  December  1st  this  filtrate  was 
active  for  B.  enteritidis. 

These  two  examples  are  sufficient  to  show  that  the  absence  of 
the  bacteriophage  is  only  apparent.  It  must  be  remembered 
that  the  ultramicrobe  is  recognized  only  by  its  activity,  and  con- 
sequently its  presence  in  a  filtrate  can  be  detected  only  by  test- 
ing it  against  a  bacterium  for  which  it  has  a  definite  activity. 
On  the  other  hand,  it  is  not  possible  to  carry  out  the  examina- 
tion on  strains  of  all  bacteria  which  may  be  found  normally  or 
occasionally  in  the  intestine.  For  this  there  are  several  reasons, 
the  chief  being  the  difficulty  of  numbers,  for  there  is  no  species 
of  bacteria  which  may  not  be  found  in  the  intestinal  tract  at  one 
time  or  another.  We  have  seen  further  that  certain  bacterial 
species  are  not  "  homogeneous"  as  regards  the  bacteriophage. 
B.  coli  is  of  this  group.  Certain  strains  are  attacked  while 
others  remain  unharmed.  It  would  then  be  necessary  to  make 
tests  with  all  varieties  of  a  single  species;  a  new  impossibility. 
Finally,  it  is  known  that  there  exist  in  the  intestine  certain  bac- 
teria, revealed  by  the  microscope,  which  it  is  impossible  to  iso- 
late and  cultivate.  May  the  bacteriophage  not  live  in  commensal- 
ism  with  these  bacteria,  with  which  they  may  form  in  the  in- 
testine ''mixed  cultures?"  It  may  even  be  that  the  impossibility 


THE   BACTERIOPHAGE   IN   THE   HEALTHY   INDIVIDUAL 


231 


of  isolating  these  bacteria  may  be  due  solely  to  this  phenomenon 
of  commensalism,  for  we  have  seen  that  the  agar  on  which  mixed 
cultures  are  planted  sometimes  remains  free  of  bacterial  colonies. 


TABLE  1 


DATE 

VIRULENCE  OF  THE  INTESTINAL  BACTERIOPHAGE  FOR 

B.  coli 

B.  dysenteriae 

Bacillus 

Other  organisms 

.1 

2 

to 

Flexner 

1 

Typhosus 

<J 

09 

1 

A 

January  15 

0 

+ 

0 
0 

+ 
+ 
+++ 
+ 
+ 

0 

+ 

0 

+++ 
+ 
+++ 
+++ 
+ 
+ 
+ 
+ 
+ 

0 

+ 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

+ 
+++ 

0 

+++ 

0 

+++ 

0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

++ 

0 
0 
0 
0 
0 
0 
0 

++ 

0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

+++ 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

++ 

0 
0 

0 
0 
0 

+++ 

0 
0 
0 

++ 

0 
0 
0 
0 
0 
0 
0 
0 
0 

+++ 

0 
0 
0 
0 
0 

Salmonella-f+  + 
B.  enteritidis+  + 

February  1  
February  15  .... 
March  1  . 

March  15 

April  1  

April  15      ... 

May  1  

May  15  

June  1 

June  15  

July  1  

July  15  ... 

August  1 

August  15  

September  1  .... 
September  15  ... 
October  1  

October  15  
November  1  .... 
November  15  ... 
December  1  
December  15.  ... 

However  this  may  be,  the  table  here  given  shows  the  results 
of  the  tests  made  on  the  normal  man  in  question.  The  examina- 
tions are  adequate  to  show  that  the  bacteriophage  is  a  normal 
inhabitant  of  the  intestine  (table  1). 

During  the  course  of  this  same  year  this  individual  showed  at 
two  different  times,  July  3,  and  September  26,  slight  intestinal 


UNIVERSITY  OF  CALIFORNIA 
0CPABTMENT  OF  CIVIL  ENGINEERING 


232 


THE   BACTERIOPHAGE 


disturbances  lasting  some  hours.  The  first  time  there  was  no 
obvious  cause;  the  second  attack  followed  a  suspected  meal  taken 
in  a  village  tavern.  On  each  occasion  specimens  of  the  stools 
were  examined  on  the  following  days.  The  results  are  recorded 
in  table  2. 

There  can  be  no  doubt  that  in  the  first  case  the  cause  was 
B.  dysenteriae  Flexner,  and  in  the  second  B.  paratyphosus  B. 
Disease  was  aborted,  the  bacteriophage  having  quickly  acquired 
virulence  for  the  invading  germ. 

TABLE  2 


DATE 

B.  COLI 

B.  DYSENTERIAE 

BACILLUS 

Shiga 

Flex- 
ner 

Hiss 

Typho- 

8  US 

Para  A 

ParaB 

July  4 

++  + 

0 

0 
0 

0 
0 
0 
0 
0 

0 

0 
0 
0 
0 
0 
0 

0 
0 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 

0 

July  5  

July  6 

July  7.  .  . 

July  8 

September  27  

September  28  .. 

September  29  

September  30  

October  1 

October  2  

October  3 

We  will  complete  this  section  by  giving  the  results  of  tests 
made  on  the  stools  of  three  normal  persons  (aged  39,  22,  and  17 
years)  during  the  period  of  the  15th  to  the  30th  of  July.  Only 
the  bacteria  against  which  the  bacteriophage  showed  a  virulence 
in  each  of  the  three  persons  designated  by  the  numbers  I,  II,  and 
III,  are  recorded  (table  3). 

It  is  unnecessary  to  multiply  examples.  All  of  the  experi- 
ments which  have  been  conducted  have  given  comparable  results. 
One  conclusion  stands  out;  the  bacteriophage  is  a  normal  in- 
habitant of  the  human  intestinal  canal. 


THE   BACTERIOPHAGE   IN   THE   HEALTHY   INDIVIDUAL 


233 


THE   BACTERIOPHAGE   IN  THE  HORSE 

Sixty-two  specimens  of  manure  derived  from  horses  living 
both  in  cities  and  in  the  country,  in  France  and  in  Indo-China, 
have  been  examined  and  all  contained  an  active  bacteriophage. 
A  list  of  the  animals  is  given,  and  the  results  of  the  examination 
are  recorded  in  table  4. 

No.  1.  Horse  No.  21  of  the  Pasteur  Institute.  This  horse 
was  used  in  the  production  of  anti-dysentery  serum.  The  ex- 
amination was  made  three  days  after  the  injection  of  Shiga  toxin. 

TABLE  3 


DATE 

i 

II 

III 

July  15 

c+ 

C+-4--4- 

o 

July  16 

C+ 

C+++ 

C+++ 

July  17  

o 

c+ 

o 

July  18  

C+++  H+++ 

o 

o 

July  19  

C+++H++ 

o 

C++  B+ 

July  20  

C++  H+++ 

o 

C+  B+ 

July  21    . 

C++  H+ 

c+ 

o 

July  22  

0 

o 

o 

July  23  

o 

o 

c+ 

July  24  .  . 

C+++ 

o 

o 

July  25  

C+  + 

o 

o 

July  26  

o 

o 

o 

July  27.  .  . 

C++  Sh'+++ 

C++ 

C++4- 

July  28  . 

C+  Sh  + 

o 

o 

July  29  

o 

c+ 

o 

July  30  

c+ 

c+ 

o 

C  =  B.  coli;    H  =  B.  dysenteriae  Hiss;  Sh.  =  B.  dysenteriae  Shiga; 
B  =  B.  paratyphosus  B. 

No.  2.    Horse  No.  21  (above),  tested  ten  days  later. 

No.  3.  Horse  No.  21  (above),  tested  four  months  later,  the 
examination  being  made  48  hours  after  a  toxin  injection. 

No.  4.  Horse  No.  114.  Used  in  the  production  of  Shiga  anti- 
dysentery  serum. 

No.  5.  Horse  No.  18.  Used  in  the  production  of  Shiga  anti- 
dysentery  serum. 

No.  6.  Horse  No.  18.  (above)  tested  four  months  later. 
The  specimen  was  collected  48  hours  after  the  injection  of  Shiga 
toxin. 


234 


THE   BACTERIOPHAGE 


No.  7.  Horse  No.  64.  This  horse  had  received  injections 
of  atoxic  dysentery  bacilli — Flexner  and  Hiss — for  two  years. 

No.  8.  Horse  No.  65.  This  horse  had  received  injections  of 
atoxic  dysentery  bacilli — Flexner  and  Hiss — for  two  years. 


TABLE  4 


HORSE 

B.COLI 

B.  DY8ENTEBIAE 

BACILLUS 

B.GALLI- 

NAKUM 

Shiga 

Flexner 

Hiss 

Typhosus 

Para  A 

ParaB 

1 

4-4- 

++++ 

+  + 

+  + 

0 

0 

0 

— 

2 

4- 

4- 

+ 

0 

0 

0 

0 

— 

3 

+ 

4-4- 

0 

0 

++ 

+ 

+ 

— 

4 

4-f 

4-4- 

+ 

-h 

0 

+ 

++ 

0 

5 

4-4- 

4-4-4- 

+++ 

++'+ 

0 

0 

0 

0 

6 

+ 

0 

+++ 

+++ 

0 

0 

0 

0 

7 

4-4- 

+4-4-4- 

++++ 

++ 

0 

0 

0 

— 

8 

4~t- 

+ 

++++ 

++++ 

0 

0 

0 

— 

9 

+  + 

4-4-4-4- 

++ 

++ 

0 

0 

0 

— 

10 

4~t- 

0 

++ 

++ 

0 

0 

+ 

— 

11 

4- 

0 

+ 

++ 

0 

0 

0 

0 

12 

+  + 

4-4-4- 

+ 

++ 

0 

0 

4- 

0 

13 

4"f 

4-4- 

++ 

+ 

+ 

0 

0 

0 

14 

4~h 

++++ 

++ 

++ 

0 

0 

0 

0 

15 

0 

4-4-4-+ 

++ 

+++ 

0 

0 

0 

0 

16 

4-4- 

4-+++ 

++++ 

++++ 

++ 

+++ 

+  4- 

+  4- 

17 

4-4- 

+++ 

++ 

++ 

•  + 

++ 

0 

4-4- 

18 

+++ 

++ 

+++ 

+++ 

+++ 

+++ 

4-4- 

4-4-4- 

19 

++ 

++++ 

+ 

+ 

0 

0 

4-4- 

0 

20 

0 

++ 

0 

0 

0 

0 

0 

0 

21 

4- 

+ 

+ 

0 

0 

4- 

4-4- 

0 

22 

4-4- 

0 

0 

0 

0 

0 

4-4- 

0 

23 

4-4- 

+++ 

++ 

++ 

0 

0 

4-4- 

0 

24 

4- 

++++ 

+++ 

++ 

++ 

0 

4- 

0 

25 

4- 

++ 

0 

0 

0 

0 

0 

0 

26 

4-4-4- 

+++ 

++ 

++ 

0 

0 

+++ 

0 

No.  9.     Horse  No.  68.     This  horse  had  received  injections  of 
atoxic  dysentery  bacilli — Flexner  and  Hiss — for  two  years. 
No.  10.     This  horse  was  receiving  injections  of  B.  anthracis. 
No.  11.     This  horse  was  receiving  injections  of  B.   anthracis. 
No.  12.     A  carriage  horse  in  Paris. 
No.  13.     A  carriage  horse  in  Paris. 


THE  BACTEEIOPHAGE   IN  THE   HEALTHY  INDIVIDUAL         235 

No.  14.    A  carriage  horse  in  Paris. 

No.  15.  The  same  animal  as  No.  14  (above)  but  tested  four 
days  later. 

No.  16.  A  farm  horse  on  a  farm  where  avian  typhosis  was 
present. 

No.  17.  A  farm  horse  on  a  farm  where  avian  typhosis  was 
present. 

No.  18.  A  farm  horse  on  a  farm  where  avian  typhosis  was 
present. 

No.  19.     A  race  horse  at  Chantilly. 

No.  20.    A  race  horse  at  Chantilly. 

No.  21.  The  same  animal  as  No.  19  (above)  but  tested  eight 
days  later. 

No.  22.  The  same  animal  as  No.  20  (above)  but  tested  eight 
days  later. 

No.  23.    A  carriage  horse  at  Saigon. 

No.  24.    A  carriage  horse  at  Saigon. 

No.  25.    A  saddle-horse  at  Nha-Trang  (Annam). 

No.  26.    A  saddle-horse  at  Phantiet  (Annam). 

There  is  no  point  in  adding  to  this  list;  the  thirty-six  other 
specimens  gave  entirely  comparable  results. 

Incidentally,  horses  No.  19  and  No.  20,  were  examined  to  see 
if  the  bacteriophage  presented  a  virulence  for  various  other  bac- 
teria, including  the  following  organisms: 

1.  A  cocco-bacillus   (?)  isolated  from  the  nasal  mucus  of  a 
horse  in  the  same  stable  which  showed  the  evening  before  an 
elevation  of  temperature: 

Horse  No.   19  (++),  horse  No.  20  (++)• 

2.  A  cocco-bacillus  isolated  by  Cesari  from  the  blood  of  a 
horse  slaughtered  in  the  abattoir  of  Vaugirard: 

Horse  No.  19  (+)  horse  No.  20  (++). 

3.  Salmonella  (hog  cholera): 

Horse  No.  19  (++),  horse  No.  20  (++). 

4.  B.  enteritidis:  Horse  No.  19  (0),  horse  No.  20  (+). 

These  results  show  that  at  a  single  time  the  bacteriophage 
may  show  a  virulence  for  a  large  number  of  bacteria.  It  is  sig- 
nificant that  only  in  horses  Nos.  16,  17,  and  18,  which  lived  in 
an  environment  contaminated  by  B.  gallinarum,  did  the  intestinal 
bacteriophage  show  a  definite  virulence  for  this  bacterium. 


236  THE   BACTERIOPHAGE 

Examination  was  made  of  twenty-three  specimens  of  serum, 
of  clot  remaining  after  the  decantation  of  the  serum,  and  of  the 
leucocytic  layer  on  top  of  this  clot,  taken  from  horses  harboring 
in  their  intestines  a  bacteriophage  active  for  B.  dysenteriae.  In 
no  case  was  a  bacteriophage  found.  In  all  instances  the  speci- 
mens of  blood  were  collected  about  two  weeks  after  the  last 
injection  of  toxin  or  of  bacilli.  All  the  specimens  of  blood  ex- 
amined came  from  horses  furnishing  anti-dysentery  serum.  It 
was  therefore  not  determined  whether  the  bacteriophage  may  not 
pass  into  the  circulation  immediately  after  the  injection,  espe- 
cially when  living  bacteria  are  used.  As  a  matter  of  fact  the  pas- 
sage of  the  intestinal  bacteriophage  into  the  circulation  has  been 
observed  in  the  rat,  and  in  the  fowl  in  cases  of  septicemia.  In 
all  cases  the  demonstration  of  the  presence,  it  might  be  said  con- 
stant presence,  in  the  excreta  of  the  horse  of  a  bacteriophage  active 
for  the  Shiga  bacillus,  and  the  absence  of  this  bacteriophage  in 
the  blood,  shows  in  an  unquestionable  manner  that  the  intestine 
is  the  only  locality  where  the  bacteriophage  normally  grows. 
This  fact  alone  is  sufficient  to  demonstrate  the  error  of  the  con- 
ception of  Bordet,  who  has  advanced  the  hypothesis  that  the 
bacteriophage  is  of  leucocytic  origin. 

THE   BACTERIOPHAGE  IN    THE   FOWL 

I  have  made  seventy  examinations  of  the  excreta  of  fowls,  and 
have  tested  the  bacteriophage  for  virulence  against  the  eight 
bacterial  strains  selected.  It  is  needless  to  give  all  the  results 
since  they  were  all  of  the  same  nature.  As  examples,  the  results 
of  only  one  or  two  tests  in  each  lot  will  be  given  (table  5). 

Nos.  1  and  2  represent  chickens  living  in  France  in  regions 
free  of  avian  typhosis  (12  specimens  examined). 

Nos.  3  and  4  represent  healthy  fowls  living  in  regions  where 
avian  typhosis  was  present  (19  other  examinations  carried  out 
on  the  eight  test  bacteria  gave  comparable  results,  particularly 
as  regards  B.  gallinarum). 

Nos.  5  and  6  represent  chickens  which  had  recovered  from 
avian  typhosis  (4  tests  made). 

Nos.  7  and  8  represent  chickens  which  died  of  avian  typhosis 
(8  other  tests  gave  similar  results).  The  bacteriophage  was  pres- 
ent but  was  not  virulent  for  the  pathogenic  bacillus. 


THE   BACTERIOPHAGE   IN   THE   HEALTHY   INDIVIDUAL 


237 


Nos.  9  and  10  were  chickens  living  in  Cochin-China  in  regions 
free  of  both  avian  typhosis  and  barbone. 

Nos.  11  and  12  represent  chickens  living  in  Cochin-China  in 
areas  where  barbone  was  present  but  free  of  avian  typhosis  (11 
tests,  all  essentially  the  same). 

Nos.  13  and  14  were  chickens  in  France  living  in  regions  where 
avian  typhosis  was  present.2 

TABLE  5 


B.  DY8ENTERIAE 


Shiga 


Flex- 
ner 


Hiss 


T^?°~  Para  A    ParaB 


B.OALLI- 

NARUM 


BACT. 
BAR- 
BONE 


1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 


4- 


0 

+++ 


4-+++ 


THE  BACTERIOPHAGE  IN  DIVERSE  ANIMALS 

Examinations  have  been  made  of  the  excreta  of  the  following 
animals  (table  6). 

No.  1.    A  monkey,  confined  in  a  cage  in  Paris. 

Nos.  2  and  3.     Cats  in  Paris. 

Nos.  4  and  5.  Cattle  living  on  a  farm  where  avian  typhosis 
was  present. 

2 1  would  recommend  that  bacteriologists  desiring  to  procure  strains  of 
the  bacteriophage  investigate  principally  the  excreta  of  horses  and 
chickens,  particularly  at  the  beginning  of  their  work.  It  is  from  these 
animals  that  it  is  most  easy  to  isolate  strains  of  the  bacteriophage  having 
a  high  activity  when  taken  from  the  body.  These  excreta  are,  moreover, 
more  readily  procured  than  the  feces  of  convalescents. 


238 


THE   BACTERIOPHAGE 


Nos.  6  and  7.  Cattle  in  France,  in  a  region  free  of  epizootic 
diseases. 

Nos.  8  and  9.  Steers  in  Cochin-China,  living  in  regions  free 

of  epizootics  (42  other  comparable  tests). 


TABLE  6 


NUMBER 

B.  COLI 

B.  DY8ENTERIAE 

BACILLUS 

BAC- 
TERIUM 
OP 
BAR- 
BONE 

B. 
GALLI- 
NARUM 

Shiga 

Flexner 

Hiss 

Typho- 

SU8 

Para  A, 

ParaB 

1 

+ 

4-4- 

0 

0 

0 

0 

0 

_ 

_ 

2 

0 

+ 

+4- 

4- 

0 

0 

0 

— 

— 

3 

+ 

0 

4- 

4- 

0 

0 

0 

— 

— 

4 

0 

0 

0 

4-4-4- 

0 

0 

0 

— 

+ 

5 

++ 

4-4- 

4- 

4- 

0 

0 

0 

— 

++ 

6 

+ 

4-4- 

4-4- 

0 

0 

0 

0 

— 

0 

7 

0 

4-4- 

0 

0 

0 

0 

0 

_ 

0 

8 

++ 

+++ 

4-4- 

4- 

0 

0 

++ 

— 

0 

9 

+ 

0 

4- 

0 

0 

0 

0 

— 

0 

10 

+ 

4-4-4-4- 

+4- 

0 

0 

0 

0 

0 

— 

11 

++ 

0 

4-+4- 

0 

0 

0 

0 

0 

— 

12 

+++ 

+ 

0 

0 

0 

0 

0 

++ 

— 

13 

+++ 

4-4- 

4-4- 

0 

0 

0 

0 

+++ 

— 

14 

4-4- 

4-4-4- 

4-+ 

0 

0 

0 

+ 

0 

— 

15 

4-4-4- 

+ 

+ 

0 

0 

0 

0 

0 

— 

16 

+ 

4-4-4- 

0 

4- 

0 

0 

+ 

+ 

0 

17 

4-4- 

+ 

0 

0 

0 

0 

0 

+ 

0 

18 

+ 

0 

0 

0 

+ 

0 

++ 

— 

0 

19 

+ 

4-4- 

0 

4- 

0 

0 

0 

— 

0 

20 

++ 

4-4- 

4-4-4- 

4-+4- 

0 

0 

+++ 

— 

++ 

21 

4-4-4- 

+ 

0 

4- 

+ 

+ 

++ 

— 

+ 

22 

+ 

4-4- 

4- 

4-4-+ 

0 

0 

0 

— 

— 

23 

0 

4- 

0 

0 

0 

0 

0 

— 

— 

24 

4-4- 

0 

4- 

0 

0 

0 

0 

— 

— 

25 

4-4- 

0 

0 

0 

0 

0 

0 

— 

— 

Nos.  10  and  11.  Buffaloes  living  in  regions  free  of  barbone 
(14  other  comparable  tests). 

Nos.  12  and  13.  Healthy  buffaloes  living  in  regions  where 
barbone  was  present  (24  other  comparable  tests). 

Nos.  14  and  15.  (for  comparison)  Buffaloes  sick  (14)  or  dead 
(15)  of  barbone.  Eight  other  tests  have  been  made;  five  were 


THE   BACTERIOPHAGE   IN   THE   HEALTHY   INDIVIDUAL         239 

comparable  to  those  cited.  In  three  others  a  bacteriophage  was 
not  found;  if  it  was  present  it  was  inactive  for  the  eight  test 
organisms. 

Nos.  16  and  17.     Swine  in  Cochin-China,  in  a  barbone  area. 

Nos.  18  and  19.     Swine  in  Paris. 

Nos.  20  and  21.  Swine  in  France,  on  a  farm  infected  with 
avian  typhosis  (4  other  comparable  results). 

Nos.  22  and  23.  Rabbits  living  in  cages  at  the  Pasteur  Insti- 
tute (4  other  comparable  findings). 

Nos.  24  and  25.     Goats  in  Paris. 

CONCLUSIONS 

In  brief,  in  all  the  healthy  animals  examined  the  presence  of 
a  bacteriophage  possessing  virulence  for  one  or  another  of  the 
intestinal  bacteria  selected  as  test  organisms  has  been  demonstrated. 

These  examinations  show  that  in  an  epizootic  area  the  intestinal 
bacteriophage  of  refractory  animals  is  generally  possessed  of  viru- 
lence for  the  bacterium  causing  the  epidemic;  on  the  contrary, 
this  is  never  observed  outside  of  the  foci  of  infection. 

The  activity  of  the  bacteriophagous  ultramicrobe  towards  a 
given  bacterium  can  only  be  explained  as  the  result  of  growth  at 
the  expense  of  this  bacterium.  Tests  of  the  virulence  of  the  ul- 
tramicrobe in  animals  inhabiting  an  epizootic  region  or  living  in 
an  area  free  of  the  infection,  against  the  bacterium  considered 
as  the  cause  of  the  disease,  are,  among  other  proofs,  conclusive 
in  this  respect.  The  experiments  in  vitro  are  in  accord  with  the 
facts  observed  in  animals.  All  the  facts  agree  in  showing  that 
in  the  body  the  bacteriophage  ceases  to  be  active  for  a  bacterium 
a  few  days  after  the  destruction  of  this  bacterium.  With  ani- 
mals, the  ingestion  of  typhoid,  paratyphoid,  and  especially, 
dysentery  bacilli,  must  be  extremely  frequent,  since  in  animals 
the  intestinal  bacteriophage  is,  except  in  rare  instances,  virulent 
for  one  or  another  of  these  organisms. 

The  sum  total  of  the  results  suggests  that  the  bacteriophage 
possesses  "  co-virulences"  or  "  accessory  virulences"  extending 
to  organisms  belonging  to  the  same  group  as  the  invading  bacillus. 
For  example,  a  bacteriophage  increasing  its  virulence  for  the  Shiga 
strain  of  B.  dysenteriae  must  at  the  same  time,  although  to  a  less 


240  THE   BACTEBIOPHAGE 

degree,  attack  the  bacillus  of  Flexner,  or  that  of  Hiss,  or  both  at 
once.  It  is  difficult  to  explain  in  any  other  manner  the  simul- 
taneous appearance  of  virulence  for  several  bacilli  of  the  same 
group.  These  co-virulences  extend  generally  to  the  bacillary 
species  which  show  among  themselves  the  phenomenon  of  co- 
agglutination. 

With  man,  also,  much  less  exposed  to  contagion  because  of 
his  mode  of  life,  the  activity  of  the  bacteriophagous  ultramicrobe 
for  the  typhoid,  paratyphoids,  and  dysentery  bacilli  is  very  fre- 
quent. Each  time  that  it  occurs  it  can  only  be  the  indication  of 
an  incipient  infection,  which  usually  passes  undetected.  The 
bacteriophage,  as  a  result  of  its  rapid  adaptation  destroys  the 
invading  bacilli  before  they  can  multiply.3 

The  ultramicrobial  bacteriophage  is  a  normal  inhabitant  of 
the  intestine  of  all  animals.  Furthermore,  as  we  have  seen, 
it  has  a  great  vitality.  Thanks  to  its  minuteness  it  is  able  cer- 
tainly to  filter  through  soils  which  arrest  bacteria.  Everything 
shows  that  it  must  be  extremely  widely  disseminated  in  the  ex- 
ternal world.  Everything  which  at  any  time  may  be  contami- 
nated by  the  excreta  of  any  animal  must  contain  it.  It  must  be 
particularly  abundant  where  there  are  living  organisms;  in  the 
soil,  in  rivers,  and  in  the  ocean.4  And  everywhere  it  must  be 
the  principal  factor  in  the  destruction  of  bacteria. 

3  It  seems  then,  even  in  animals  considered  refractory,  that  there  occurs 
at  times  a  delay  in  the  adaptation  of  the  bacteriophage.  This  is  noted, 
for  example,  in  cases  of  dysentery  (Shiga  bacillus)  in  horses  in  tropical 
countries. 

4 1  have  isolated  a  strain  of  the  bacteriophage  active  for  B.  coli  from  a 
specimen  of  sea-water  taken  off  from  the  estuary  of  Mekong,  and  a  second 
taken  from  the  Mediterranean  off  Marseilles.  I  was  unable  to  isolate  a 
strain  from  a  specimen  of  water  from  the  Indian  Ocean  at  a  point  approxi- 
mately 60°  East  longitude  and  10°  North  latitude.  Dumas  has  isolated 
strains  from  a  specimen  of  garden  soil  and  from  the  tap-water  in  Paris. 
Beckerich  and  Hauduroy,  at  the  Institute  of  Hygiene  at  Strasbourg,  have 
isolated  a  number  of  strains  active  for  B.  coli,  for  B.  typhosus,  and  for  dys- 
entery organisms,  from  different  specimens  of  soil,  from  the  water  of  the 
111,  and  from  Rhine  water  after  sand  filtration. 

These  findings  allow  us  to  draw  an  important  conclusion,  which  has  a 
bearing  on  hygiene.  The  bacteriophage  exercises  a  preponderant  role  in 
the  defense  of  the  organism.  It  is  therefore  of  great  interest  that  drinking 


CHAPTER  III 

IMMUNIZATION  BY  MEANS  OF  THE  BACTERIOPHAGE 

Immunization  against  Avian  Typhosis.     Immunization  against  Barbone- 
Immunization  against  Dysentery.     Conclusions. 

The  single  test  scientifically  acceptable  for  a  theory  of  im- 
munity can  be  furnished  only  by  the  reproduction  of  this  im- 
munity in  an  animal  naturally  susceptible.  That  is,  in  placing 
this  animal  by  experiment  in  the  condition  to  which  immunity 
is  attributed. 

A  strain  of  the  bacteriophage  active  for  a  given  bacterium  can 
be  isolated  and  cultivated  in  vitro  indefinitely  at  the  expense  of 
this  bacterium,  thus  maintaining  its  virulence.  In  this  way  any 
desired  amount  of  culture  can  be  obtained. 

If  the  theory  of  immunity  by  the  bacteriophage  which  we  have 
deduced  from  the  investigations  made  on  natural  disease  is 
correct  we  should  be  able  at  will  to  reproduce  all  the  phenomena 
leading  to  recovery,  provided  there  do  not  exist  at  the  time  of 
intervention  organic  lesions  incompatible  with  life.  We  should 
likewise  be  able  to  place  the  exposed  individual  in  the  same  re- 
fractory state  enjoyed  by  the  person  who  has  passed  through  the 
epidemic  period  unaffected.  It  is  for  this  reason  that  up  to  the 
present  time  I  have  confined  myself  almost  entirely  to  a  study  of 
the  role  of  the  bacteriophage  in  animals,  since  these  alone  permit 
of  experimental  confirmation. 

water  should  contain  the  greatest  possible  number  of  bacteriophagous 
ultramicrobes  virulent  for  the  agents  of  contagious  diseases.  It  is  certain 
that  to  obtain  such  waters,  containing  at  once  the  greatest  possible  number 
of  virulent  ultramicrobes  and  the  smallest  possible  number  of  bacteria  it 
is  necessary  to  use  filtered  river  water  and  not  stored  waters.  As  potable 
waters  the  first  are  at  the  same  time  superior,  from  both  the  hygienic 
and  economic  points  of  view. 

Attention  may  be  called  to  the  fact  that  in  certain  cases  a  search  for 
a  bacteriophage  virulent  for  a  given  bacterium  may  be  of  some  significance 
in  hydrological  investigations. 

241 


242  THE   BACTEBIOPHAGE 

The  immunization  experiments  by  means  of  cultures  of  the 
bacteriophage  have  been  conducted  in  two  different  ways: 

a.  In  an  epizootic  area  of  avian  typhosis,  where  there  would 
be  an  immunization  against  natural  infection;  and 

b.  In  a  non-infected  area,  as  in  barbone,  where  the  results 
could   be   checked   against   experimental   controls.     These   last 
experiments  have  shown  clearly  the  exact  conditions  under  which 
immunization  by  means  of  the  bacteriophage  can  be  carried  out. 

IMMUNIZATION   AGAINST  AVIAN   TYPHOSIS 

Further  mention  need  not  be  made  of  the  immunization  ex- 
periments conducted  in  the  laboratory,  which  have  been  dis- 
cussed in  the  chapter  dealing  with  the  phenomena  observed  in 
the  course  of  epizootics  of  avian  typhosis. 

Immunization  experiments  in  a  region  where  the  epizootic 
was  present,  as  in  the  case  of  avian  typhosis,  presented  an  especial 
difficulty,  or  rather,  a  complication.  It  has  been  mentioned 
that  aside  from  the  typical  typhosis,  due  to  B.  gallinarum,  there 
are  several  varieties  of  paratyphoses,  each  caused  by  a  particu- 
lar species  of  bacterium.  The  differences  which  these  bacterial 
species  present  from  the  biochemical  and  agglutinative  points 
of  view  and  which  serve  to  differentiate  them  are  of  no  particu- 
lar significance  from  the  point  of  view  of  this  study.  In  so  far 
as  the  action  of  the  bacteriophage  is  concerned  for  each  of  these, 
a  strain  of  bacteriophage  having  an  extreme  virulence  (+  +  +  +) 
for  B.  gallinarum  possesses  the  same  activity  for  all  the  French 
and  American  strains  as  well  as  for  B.  jeffersonii.  The  activity 
is  less  pronounced  for  B.  pullorum  A,  still  less  for  B.  pullorum  B, 
and  is  lacking  for  B.  pfaffi  and  for  B.  rettgerei.  With  such  a 
strain  of  bacteriophage  the  reactions  are:  B.  gallinarum  +  +  +  +, 
B.  jeffersonii  +  +  +  +  ,  B.  pullorum  A  ++,  B.  pullorum  B  +, 
B.  pfaffi  0,  and  B.  rettgerei  0.  Inversely,  a  strain  of  bacteriophage 
secured  from  fowls  resistant  to  paratyphosis  due  to  B.  pfaffi 
(focus  at  Trainel,  Aube)  had  the  following  virulences:  B.  galli- 
narum 0,  B.  jeffersonii  0,  B.  pullorum  A  0,  B.  pullorum  B  +, 
B.  pfaffi  +  +  +  +  ,  and  B.  rettgerei  0. 

The  immunization  experiments  thus  become  singularly  com- 
plicated, particularly  since  both  typhosis  and  the  paratyphoses 


IMMUNIZATION   BY  MEANS   OF  THE   BACTERIOPHAGE  243 

may  be  found  in  the  same  areas,  as  is  also  true  for  human  enteric 
infections.  In  routine  practise  the  solution  is  simple;  it  is  sufficient 
to  immunize  the  poultry  with  a  mixture  of  cultures  of  different 
strains  of  the  bacteriophage  active  against  the  diverse  pathogens, 
the  causes  of  typhosis  and  the  paratyphoses.  In  the  preliminary 
investigations  this  was  not  possible,  for  the  differences  be- 
tween the  different  diseases  had  not  been  recognized  when  I 
first  undertook  this  study.  The  different  bacilli,  the  agents 
of  the  paratyphoses,  had  been  studied  in  the  United  States  but 
their  simultaneous  presence  in  foci  of  typhosis  had  not  been  noted. 
And  in  so  far  as  B.  pfaffi  is  concerned,  discovered  by  Pfaff  in  an 
epizootic  in  Vienna,  it  had  not  then  been  incriminated  as  capable 
of  producing  disease  in  the  Gallinaceae.  These  facts  have  only 
been  disclosed  gradually  in  the  course  of  these  investigations. 

The  cultures  of  bacteriophage  used  in  the  immunization  ex- 
periments were  prepared  in  the  following  manner: 

A  culture  of  B.  gallinarum,  in  Martin  bouillon,  aged  nine  or 
ten  hours,  that  is,  very  young  but  showing  a  definite  turbidity, 
is  inoculated  with  a  bacteriophage  isolated  from  the  excreta  of  a 
recovered  chicken  and  possessing  a  high  virulence  for  the  patho- 
genic bacillus.  After  about  12  hours  the  bacterial  lysis  is  com- 
pletely finished  and  the  bouillon  is  perfectly  limpid.  This  cul- 
ture is  filtered  through  a  bougie1  and  distributed  into  ampoules 
which  are  sealed. 

The  dose  employed  for  immunization  has  been  in  all  cases  0.5 
cc.,  given  subcutaneously.  The  point  of  injection  is  of  no  im- 
portance for  the  slightest  local  or  general  reaction  has  never 
been  observed. 

Experiment  I.  The  following  experiments  were  conducted 
in  1919  and  1920  in  the  neighborhood  of  Agen  with  the  assistance 
of  M.  Lambert,  D.V.M. 

Barnyard  1.  The  epizootic  began  in  August,  1919.  By  October.  2,  110 
of  160  fowls  had  died.  The  50  survivors,  of  which  5  were  already  affected, 
were  inoculated  with  the  culture  of  bacteriophage.  The  5  sick  chickens 

1  We  have  seen  that  whatever  may  be  the  virulence  of  the  inoculated 
bacteriophage  one  may  always  obtain  secondary  cultures  in  a  certain  num- 
ber of  tubes.  Filtration  is  thus  essential. 


244  THE   BACTERIOPHAGE 

recovered  and  the  epizootic  stopped  abruptly  and  definitely  on  the  same 
day  as  the  immunization. 

Barnyard  2.  The  epizootic  began  on  about  August  20.  By  October  6, 
120  of  200  fowls  had  died.  The  80  survivors,  of  which  7  were  sick,  received 
an  injection  of  the  antigallinarum  bacteriophage.  The  7  recovered;  the 
epizootic  immediately  and  permanently  disappeared. 

Barnyard  3.  The  epizootic  began  October  10.  By  the  15th,  21  fowls  had 
died.  The  130  that  were  alive,  of  which  8  were  already  sick,  were  inocu- 
lated. The  8  recovered  and  the  epizootic  disappeared  from  the  day  of  the 
inoculation. 

Barnyard  4-  The  epizootic  began  on  about  November  15.  By  December 
1,  26  of  51  fowls  were  dead.  The  25  survivors,  among  which  were  4  which 
were  infected,  were  inoculated.  One  of  the  sick  animals  died,  the  other 
3  recovered.  The  mortality  stopped  from  the  date  of  the  inoculation. 

Barnyard  5.  The  epizootic  began  about  November  25.  By  December 
1,  7  of  60  chickens  had  succumbed.  The  53  survivors  were  inoculated.  Of 
these  4  were  sick.  The  sick  animals  recovered  and  no  new  cases  appeared. 

Barnyard  6.  The  epizootic  began  on  December  16.  On  the  28th,  40  of 
142  fowls  had  died.  The  102  survivors,  of  which  3  were  infected,  were  in- 
oculated. The  sick  recovered  and  the  disease  abruptly  stopped. 

Barnyard  7.  The  epizootic  began  on  January  2.  By  January  14,  15 
of  50  animals  had  died.  The  35  survivors  were  inoculated.  No  new  cases 
developed  from  this  time  on. 

Barnyard  8.  The  epizootic  began  on  about  January  15  with  a  daily 
mortality  of  4  to  6  fowls.  On  January  21  the  121  survivors,  including  5 
which  were  sick,  were  inoculated.  The  sick  recovered  and  the  epizootic 
stopped  at  once. 

Barnyard  9.  The  epizootic  began  on  about  February  10.  By  February 
20, 14  chickens  had  died  from  among  the  original  84.  The  70  survivors  were 
inoculated  and  the  disease  disappeared  at  once. 

Barnyard  10.  The  epizootic  began  about  February  25.  By  March  1, 
20  chickens  had  died.  The  120  survivors,  of  which  5  were  sick,  were  inocu- 
lated. The  5  recovered  and  the  epizootic  stopped. 

Barnyard  11.  The  epizootic  began  on  February  4.  From  February  4 
to  10, 10  chickens  died.  On  February  10  the  48  living  fowls  were  inoculated 
in  the  wing  with  0.5  cc.  of  the  antigallinarum  bacteriophage,  as  had  been 
all  the  chickens  in  the  ten  preceding  experiments.  The  epizootic  con- 
tinued its  course  and  5  chickens  died  from  February  10  to  17.  On  Feb- 
ruary 17  the  43  fowls  which  remained  were  inoculated  with  0.5  cc.  of  a  mix- 
ture of  four  strains  of  bacteriophage :  active  against  B.  gallinarum,  B.  pullo- 
rum  A,  B.  pullorum  B,  and  B.  pjaffi.  The  epizootic  stopped  immediately 
after  this  second  inoculation. 

Barnyard  12.  This  barnyard  was  adjacent  to  the  preceding  and  here  the 
same  facts  were  observed.  A  first  inoculation  made  on  February  9  on  80 
chickens  with  a  culture  of  the  antigallinarum  bacteriophage  was  without 


IMMUNIZATION   BY  MEANS  OF  THE   BACTERIOPHAGE  245 

effect.  The  epizootic  stopped  abruptly  after  an  inoculation  of  bacterio- 
phage  active  for  the  bacillary  agents  of  the  paratyphoses,  made  on  Feb- 
ruary 17. 

Examination  of  the  blood  of  fowls  dead  in  Barnyard  No.  12  resulted  in 
the  isolation  of  a  B.  pfaffi,  type  of  bacillus.  This  organism,  then,  was 
responsible  for  the  epizootics  in  groups  11  and  12.  In  this  connection  I  will 
only  mention  the  instance  of  the  epizootic  of  paratyphosis  at  Trainel  of 
which  I  have  spoken  in  the  chapter  on  avian  typhosis.  This  outbreak  was 
likewise  due  to  B.  pfaffi,  and  was  controlled  by  the  inoculation  of  an  anti- 
pfaffi  bacteriophage. 

Experiment  II.  This  was  performed  at  Foully  en  Auxois 
with  the  assistance  of  MM.  Voillot  and  Bouhier,  D.V.M. 

Barnyard  1.  On  Januarys,  20  chickens  were  taken  at  random  from 
a  poultry-yard  containing  about  100  fowls  where  typhosis  had  appeared. 
These  20  were  immunized  with  a  culture  of  anti-gallinarum  bacteriophage. 
On  February  7  the  immunized  birds  were  all  alive  and  in  perfect  condition, 
while  the  epizootic  had  continued  to  spread  among  the  non-immunized 
animals,  of  which  only  about  20  remained. 

Barnyard  2.  On  February  23  the  surviving  chickens  of  a  poultry-yard 
containing  at  that  time  102  animals  were  immunized.  The  epizootic  which 
began  about  10  days  previously,  and  which  had  resulted  in  a  daily  mortality 
of  4  or  5  chickens,  stopped  quickly  and  permanently  from  the  time  of  the 
immunization.  The  epizootic  continued,  on  the  contrary,  to  ravage  with 
the  same  intensity  as  formerly  in  all  the  neighboring  poultry-yards  which 
served  as  controls. 

Experiment  III.  This  experiment  was  conducted  at  Provins 
with  the  aid  of  M.  Sorriau  D.V.M.,  in  an  important  poultry- 
yard  where  typhosis  was  present  in  endemic  form. 

For  several  months  the  daily  mortality  had  been  2  or  3  fowls.  On  Jan- 
uary 25  the  225  survivors  were  immunized.  The  epizootic  immediately 
and  permanently  disappeared  from  the  date  of  the  immunization. 

Experiment  IV.  Performed  at  Rouillac,  Charente,  with  the 
assistance  of  M.  Chollet,  D.V.M. 

On  December  15,  100  fowls  were  immunized  in  a  poultry-yard  where 
typhosis  had  appeared  about  ten  days  previously.  The  daily  mortality 
had  been  from  4  to  6  animals.  With  the  immunization  there  was  an  imme- 
diate and  permanent  cessation  of  the  epizootic.  Typhosis  continued  to 
prevail  on  all  the  neighboring  farms.  Among  the  100  chickens  inoculated, 
about  12  were  already  affected.  Of  these  only  2  died,  2  and  3  hours  after 
the  injection. 


246  THE   BACTERIOPHAGE 

Experiment  V.  This  test  was  conducted  with  the  assistance 
of  Dr.  Ormieres  at  Carcassonne. 

The  epizootic  began  during  the  month  of  August.  By  October  1,  80 
chickens  had  succumbed.  The  120  survivors  were  immunized.  The 
epizootic  stopped  immediately  and  no  further  cases  appeared  after  the 
date  of  the  immunization. 

Experiment  VI.  This  experiment  was  conducted  with  the 
assistance  of  M.  Mesnard,  Departmental  Veterinarian  at  Angou- 
l&ne.  In  these  experiments  the  chickens  were  immunized  by 
the  ingestion,  on  bread,  of  about  1  cc.  of  an  antigallinarum 
bacteriophage. 

A.  On  July  2  the  50  chickens  surviving  in  a  poultry-yard  where  typhosis 
had  been  prevalent  for  six  weeks,  with  a  daily  mortality  of  2  or  3  fowls, 
each  ingested  about  1  cc.  of  the  bacteriophage  culture.  Seven  months 
later  no  new  case  had  developed  since  the  time  of  the  ingestion. 

B.  The  same  test  was  performed  on  October  15  on  about  100  chickens  on  a 
neighboring  farm  where  typhosis  had  been  present  fro  several  months. 
The  epizootic  was  immediately  and  completely  checked. 

In  both  of  these  cases  the  disease  continued  to  spread  throughout  the 
neighboring  poultry-yards  that  were  held  as  controls. 

It  appears  needless  to  multiply  such  examples.  In  all  cases 
the  picture  has  been  the  same.  The  epizootic  disappeared  from 
the  time  that  the  culture  of  bacteriophage  virulent  for  the  patho- 
genic bacterium,  the  cause  of  the  epizootic,  had  been  introduced 
into  the  organism  of  the  susceptible  animal,  whether  this  was 
brought  about  by  injection  or  ingestion.  We  will  see  later  that 
this  last  mode  of  administration  is  somewhat  less  efficient  than 
injection. 

On  the  contrary,  injections  of  cultures  of  a  bacteriophage 
active  for  B.  gallinarum,  the  specific  cause  of  typhosis,  had  in 
general,  no  effect  when  the  epizootic  was  a  paratyphosis,  par- 
ticularly in  the  case  of  infections  due  to  B.  pfaffii.  In  practise, 
it  is  only  necessary  to  inject  a  mixture  of  different  strains  of 
bacteriophage  active  for  the  various  pathogenic  bacteria  that 
may  produce  the  epizootic.  This  mixture  should  also  include  a 
strain  active  for  chicken  cholera.  It  will  be  very  easy  to  accom- 
plish this,  for  the  dose  of  0.5  cc.  which  I  have  arbitrarily  adopted 


IMMUNIZATION  BY  MEANS  OF  THE   BACTEKIOPHAGE  247 

is  indeed  much  larger  than  necessary,  as  we  will  see.  Even  in 
mixing  five  or  six  different  strains  of  bacteriophage,  the  quantity 
necessary  to  effect  immunization  is  not  more  than  a  fraction  of 
a  cubic  centimeter. 

In  the  course  of  the  experiments  cited  there  has  been  no 
selection.  All  of  the  animals  of  the  poultry-yard,  even  though 
they  were  moribund,  received  the  immunizing  injection.  About 
100  sick  chickens  have  therefore  been  injected,  and  the  mortality 
among  these  has  been  5  per  cent.  This  is  an  appreciable  reduc- 
tion since  the  mortality  among  affected  animals  varies  from  one 
hundred  per  cent  at  the  beginning  of  the  epizootic  to  95  per  cent, 
when,  after  some  weeks,  the  disease  appears  in  only  sporadic 
cases. 

A  culture  of  the  bacteriophage,  as  we  have  shown  in  several 
ways,  is  composed  of  bacteriophagous  ultramicrobes  suspended 
in  a  medium  containing  the  dissolved  bacterial  substance;  the 
bacteria  which  have  been  destroyed  by  the  action  of  the  lysins 
secreted  by  the  ultramicrobe.  What,  among  these  different 
principles,  is  the  one  which  plays  the  active  role  in  the  protec- 
tion of  the  healthy  animal  or  in  the  one  already  sick,  under  the 
conditions  of  the  experiment,  that  is,  in  a  contaminated  area? 
Unquestionably  it  is  the  bacteriophagous  germs  themselves.  The 
immediate  protection  assured  by  the  injection  or  even  by  the 
ingestion  of  the  bacteriophage  culture  suffices  to  demonstrate 
this.  An  organic  immunity  necessarily  requires  a  certain  time 
for  its  development.  Other  phenomena  of  immunity,  organic 
in  nature,  are  produced  only  after  an  incubation  period,  as  the 
experiments  on  barbone  will  show. 

For  the  moment,  let  us  conclude  only  that  with  sensitive 
animals  immunized  by  the  injection  of  a  culture  of  the  bacterio- 
phage active  for  the  causative  pathogenic  bacterium,  in  a  con- 
taminated area,  that  is  to  say,  in  an  area  where  frequent  reinfec- 
tions may  take  place  as  a  result  of  the  dissemination  of  the 
pathogenic  bacteria  in  the  external  environment,  the  principal 
role  of  protection  is  played  by  the  bacteriophage  itself.  The 
other  phenomena  of  immunity  which  may  later  develop,  stimu- 
lated by  the  other  substances  contained  in  the  cultures  injected, 
play  no  role  under  such  conditions,  unless  it  be  a  very  secondary 


248  THE   BACTERIOPHAGE 

role.     We  will  see  that  this  proposition  becomes  reversed  when 
similar  experiments  are  carried  out  in  a  non-contaminated  area. 

IMMUNIZATION   AGAINST    BARBONE2 

Thanks  to  the  liberality  of  the  Government  of  Cochin-China, 
which  placed  at  our  disposal  all  the  animals,  steers  and  buffaloes, 
which  we  needed,  we  have  been  able  to  study  in  detail  certain  of 
the  conditions  underlying  immunization  by  means  of  the  bac- 
teriophage.  Barbone  is,  indeed,  an  ideal  disease  for  a  study  of 
this  type.  The  blood  taken  from  an  animal  about  to  die  of  the 
disease  can  be  preserved  in  sealed  ampoules  for  at  least  six  months 
without  any  loss  in  the  virulence  of  the  bacteria  present.  Bouil- 
lon inoculated  with  a  drop  of  this  blood  yields  a  culture  which 
regularly  kills  the  steer  or  the  buffalo  in  a  dose  of  0.0002  cc. 
With  half  this  dose,  0.0001  cc.,  usually  one  out  of  two  animals 
will  be  killed.  Experimental  infection  reproduces  the  spontaneous 
disease  in  the  most  minute  details;  the  same  temperature  curve, 
the  same  symptoms,  the  characteristic  edema  at  the  point  of 
entrance  of  the  virus.  Like  the  natural  infection,  the  disease 
is  fatal;  all  animals  succumb  and  death  occurs  in  the  same  length 
of  time  in  the  two  cases,  within  twelve  to  eighteen  hours  of  the 
appearance  of  the  first  symptoms.  The  lesions  to  be  found  at 
autopsy  are  identical.  Immunization  experiments  conducted 
with  such  a  disease  provide,  then,  absolute  results. 

1  may  state  here,  once  for  all,  that  each  time  that  the  immunity 
of  one  animal  has  been  tested  by  the  inoculation  of  a  culture  of 
the  bacterium  of  barbone  this  test  has  been  controlled  by  the 
injection  of  an  equal  dose  into  a  control  animal  of  the  same  weight, 
and  never  has  the  control  resisted.     Furthermore,  although  there 
can  be  no  possible  doubt  concerning  the  cause  of  death,  confirma- 
tion has  always  been  made  by  microscopic  examination,  by  blood 
culture,  and  by  the  demonstration  of  the  lesions  at  autopsy.    The 
temperature  of  the  experimental  animals  was  taken  regularly, 
morning  and  evening,  and  the  slightest  reaction  in  the  immu- 
nized animals  could  not  have  passed  unobserved. 

2  These  experiments  were  conducted  at  Saigon  with  the  assistance  of 
G.  Le  Louet,  Chief  of  the  Veterinary  Service  in  Cochin-China. 


IMMUNIZATION   BY  MEANS   OF  THE   BACTERIOPHAGE  249 

The  strain  of  bacteriophage  employed  for  the  preparation  of 
the  cultures  destined  for  use  in  the  immunization  experiments 
had  been  isolated  from  the  feces  of  a  buffalo  which  had  passed 
unaffected  through  the  epizootic  mentioned  in  the  preceding 
chapter.  This  bacteriophage  possessed,  when  derived  from  the 
organism,  a  strong  virulence  (+  +  +)  for  the  bacterium  of  bar- 
bone.  After  about  ten  passages  in  vitro  the  virulence  became 
extreme  (+  +  +  +),  and  at  this  time  it  was  used. 

A  fairly  turbid  bouillon  culture  of  the  bacterium  of  barbone 
about  12  hours  old  received  one  drop  of  the  previously  described 
active  (+  +  +  +)  culture  of  anti-barbone  bacteriophage.  After 
about  12  hours  the  medium  became  perfectly  limpid.  This 
culture  was  filtered  through  a  Chamberlanr!  filter  (L3)  and  dis- 
tributed into  ampoules,  which  were  sealed.  I  would  call  atten- 
tion to  the  necessity  of  employing  only  cultures  with  which  the 
lysis  of  the  bacteria  has  been  complete.  Such  cultures  ought, 
moreover,  to  be  filtered  because  of  the  fact  th!at  a  secondary 
culture  may  develop  in  some  of  the  tubes. 

The  cultures  of  anti-barbone  bacteriophage  have  been  used 
after  a  variable  length  of  time, — from  twenty  days  to  five  months 
after  their  preparation.  No  difference  nas  ever  been  observed 
in  their  mode  of  action,  whatever  the  time  elapsed  between  the 
date  of  preparation  and  the  time  of  use. 

All  of  the  experiments,  except  those  dealing  with  the  effect  of 
the  age  of  the  animal  upon  the  development  of  immunity,  have 
been  effected  on  steers  of  the  indigenous  race,  in  a  perfect  state 
of  health,  aged  from  twelve  to  eighteen  months,  and  of  an  average 
weight  of  100  kgms.,3  and  on  buffaloes  aged  from  one  to  twelve 
years.  The  bovine  race  and  the  buffalo  are  equally  susceptible 
to  barbone.  In  Egypt,  Piot  has  seen  the  herds  of  cattle  deci- 
mated to  the  same  extent  as  the  herds  of  buffalo.  According 
to  our  observations  the  buffalo  may  be  rather  easier  to  immunize 
than  cattle. 

Let  us  consider  first  the  experiments  conducted  for  the  purpose 
of  determining  what  conditions  control  the  development  of  the 
immunity  resulting  from  the  injection  of  a  culture  of  the  bacterio- 

3  The  race  in  Indo-China  is  of  small  size. 


250  THE  BACTERIOPHAGE 

phage.  The  size  of  the  dose  and  the  age  of  the  animals  are  the 
two  principal  factors  whose  variation  has  the  greatest  influence 
on  the  result.  To  facilitate  discussion,  we  may  consider  the  effects 
of  smaller  and  smaller  doses,  although  in  reality  the  chronologi- 
cal order  of  the  experiments  was  somewhat  different,  since  the 
tests  were  first  made  with  the  injection  of  a  dose  arbitrarily  fixed 
at  five  cubic  centimeters.  In  this  experiment  the  animals  all 
died,  when  the  test  injection  was  given  twenty  days  later.  Think- 
ing that  the  immunizing  dose  was  inadequate  it  was  increased 
in  the  next  test  to  twenty  cubic  centimeters.  Here  again,  the 
results  were  the  same.  It  was  only  somewhat  later,  when  smaller 
doses  were  employed,  that  the  treatment  proved  to  be  efficacious. 
We  have  seen  already  that  immunization  by  means  of  bacterio- 
phage  cultures  presents  individual  peculiarities. 

Determination  of  the  immunizing  dose 

I.  Eight  steers  received  20  cc.  of  the  bacteriophage  culture  subcutane- 
ously.    Six  of  these  were  tested  after  a  lapse  of  time  varying  from  fifteen 
to  forty  days  by  the  inoculation  of  a  quantity  of  barbone  culture  repre- 
senting certainly  50  fatal  doses.    All  died  in  the  same  length  of  time  as  the 
control  animals.    The  remaining  2  were  tested,  also  with  50  fatal  doses, 
sixty  days  after  the  immunizing  injection.     They  showed  no  obvious 
disturbance.    The  two  controls  died  in  nineteen  and  twenty-two  hours 
after  the  inoculation  of  virulent  material. 

II.  Four  steers  received,  subcutaneously,  5  cc.  of  the  bacteriophage  cul- 
ture.   Three  were  tested  after  thirteen,  fifteen  and  twenty-eight  days  by 
the  inoculation  of  50  lethal  doses  of  virulent  bacilli.    All  died  in  the  same 
length  of  time  as  the  controls.    The  fourth  was  tested  on  the  fortieth  day. 
It  showed  no  reaction.     The  control  died  in  twenty-two  hours. 

III.  Forty-one   animals;   25  steers,  4  buffaloes  aged  from  one  to  two 
years,  and  12  adult  buffaloes,  received  an  injection  of  0.25  cc.  of  the  bacteri- 
ophage culture. 

A.  Eight  steers  were  tested  between  the  third  and  twelfth  days  following 
the  injection  by  the  inoculation  of  virulent  culture,  representing,  according 
to  the  weight  of  the  animal,  from  5  to  1000  surely  fatal  doses.    All  died. 

B.  Twelve  steers  and  one  buffalo  were  tested  between  the  13th  and  20th 
days,  all  by  the  inoculation  of  1000  surely  fatal  doses  of  barbone  culture. 
Five  resisted,  the  others  succumbed.     The  experiment  is  given  in  detail: 


IMMUNIZATION   BY  MEANS   OF  THE   BACTERIOPHAGE 


251 


ANIMAL 

TEST  INJECTION 
1000  FATAL 
DOSES  GIVEN 
AFTER 

RESULT 

days 

Buffalo 

13 

Eesisted  without  showing  any  reaction 

Steer  no.  50 

15 

Died  26  hours  after  the  inoculation 

Steer  no.  52 

15 

Died  20  hours  after  the  inoculation 

Steer  no.  53 

15 

Died  23  hours  after  the  inoculation 

Steer  no.  55 

15 

Died  26  hours  after  the  inoculation 

Steer  no.  56 

15 

Died  25  hours  after  the  inoculation 

Steer  no.  38 

15 

Resisted,  without  showing  any  symptoms 

Steer  no.  27 

16 

Died  68  hours  after  the  inoculation 

Steer  no.  20 

16 

Resisted,   without  showing  any  symptoms 

Steer  no.  28 

17 

Resisted,  without  showing  any  symptoms 

Steer  no.  30 

17 

Resisted,   without  showing  any  symptoms 

Steer  no.  89 

17 

Died  34  hours  after  the  inoculation 

Steer  no.  90 

17 

Died  32  hours  after  the  inoculation 

Two  control  steers  died  in  22  and  26  hours  after  the  inoculation,  and  one 
buffalo,  as  control,  died  in  19  hours. 

C.  Twenty  animals;  5  steers,  3  young  buffaloes,  and  12  adult  buffaloes 
were  tested  during  the  period  from  the  twenty-first  to  the  sixtieth  day  after 
the  immunizing  injection.  All  received  1000  surely  fatal  doses  of  culture. 
All  resisted  without  showing  any  reaction.  Five  control  animals  died, 
all  between  16  and  23  hours  after  the  inoculation  of  culture. 

IV.  Eight  steers  received  0.04  cc.  of  bacteriophage  culture.  They 
\vere  tested  after  a  variable  number  of  days  by  the  inoculation  of  5  surely 
fatal  doses  of  virulent  culture.  The  results  were : 


ANIMAL 

TESTED 
AFTER 

RESULT 

days 

Steer  no.  107 

1 

Resisted,  no  reaction  whatever 

Steer  no.  103 

1 

Resisted,  no  reaction 

Steer  no.  106 

2 

Died  36  hours  after  the  inoculation 

Steer  no.  101 

3 

Died  28  hours  after  the  inoculation 

Steer  no.    83 

4 

Resisted,  no  reaction 

Steer  no.    84 

4 

Resisted,  no  reaction 

Steer  no.  104 

5 

Resisted,  no  reaction 

The  last  steer,  No.  102,  was  tested  60  days  after  the  injection  of  the  immun- 
izing dose  by  the  inoculation  of  50  surely  fatal  doses  of  culture.  It  resisted 
without  showing  any  disturbance. 

V.  A  last  experiment,  as  a  control,  was  performed  with  a  view  to  testing 
the  practical  application  of  immunization  of  buffaloes  against  barbone  by 
M.  Le  Louet  after  my  departure  from  Saigon.  Twelve  steers  received  by 


252  THE   BACTERIOPHAGE 

subcutaneous  injection  0.25  cc.  of  bacteriophage  culture.  They  were 
tested  25  days  later  by  the  inoculation  of  2000  surely  fatal  doses  of  barbone 
culture.  They  resisted  without  showing  the  slightest  reaction.  The 
controls  died  in  from  18  to  22  hours  after  the  inoculation. 

The  injection  of  the  bacteriophage  did  not  produce  in  any  of 
the  animals,  even  in  twenty  cubic  centimeter  doses,  the  slightest 
reaction,  either  local  or  general.  The  temperature  curve  follow- 
ing the  immunizing  injection  could  be  superimposed  throughout 
on  the  curves  of  normal  untreated  animals.  From  this  it  is  clear 
that,  contrary  to  general  belief,  an  immunity  bordering  on  the 
refractory  state  may  be  acquired  without  the  manifestation  of 
the  slightest  reaction. 

During  the  course  of  these  experiments  aphthous  fever  made 
its  appearance  at  Saigon.  The  animals  in  the  course  of  immuniza- 
tion contracted  it  but  this  complication  in  no  instance  exerted 
any  influence  upon  the  development  of  immunity  to  barbone. 

From  these  different  experiments  it  may  be  deduced  that  with 
a  large  dose  of  bacteriophage  culture  the  immunity  is  slow  in 
being  established;  about  forty  to  sixty  days  with  a  dose  of  twenty 
cubic  centimeters,  more  than  twenty-eight  days  with  5  cc.  With 
0.25  cc.  it  is  not  effective  for  all  animals  until  about  the  twentieth 
day.  It  then  permits  them  to  resist  without  apparent  discomfort 
two  thousand  surely  fatal  doses  of  the  culture  of  barbone,  that 
is  to  say,  the  immunity  conferred  borders  on  the  refractory  state. 
With  the  minimal  dose  of  0.04  cc.,  or  less  than  a  normal  drop,  a 
solid  immunity  is  acquired  by  the  fourth  day.  We  are  not  con- 
cerned for  the  moment  with  the  steers  which  have  resisted  after 
twenty-four  hours;  the  immunity  which  they  enjoy  is  of  a  differ- 
ent order,  as  we  will  see  later. 

We  have  seen  in  Part  I  of  this  text  that  the  serum  of  rabbits 
which  have  received  four  injections  of  bacteriophage  culture 
possesses  the  property  of  sensitizing  the  animals  against  the  bac- 
teria for  which  the  bacteriophage  injected  was  active.  The  de- 
lay in  the  establishment  of  the  immunity  as  a  result  of  the  in- 
jection of  large  doses  of  antibarbone  bacteriophage  culture  ought 
to  induce  this  same  phenomenon.  The  injection  produces  in 
the  animals  two  phenomena  of  different  orders:  an  immunity 
and  a  sensitization  which  varies  in  intensity  according  to  the 


IMMUNIZATION   BY  MEANS   OF  THE  BACTERIOPHAGE  253 

dose  inoculated.  With  a  small  dose  the  first  surpasses  the  second 
which  disappears  quickly;  with  a  large  dose,  on  the  contrary, 
the  inhibitive  action  persists  for  a  very  long  time — about  sixty 
days  for  an  injection  of  20  cc.  As  we  have  seen  in  the  rabbit  the 
sensitization  dominates  and  persists  if  the  injections  of  the  bac- 
teriophage  are  repeated.4 

The  experiments  further  show  that  the  immunity  conferred 
by  the  injection  of  cultures  of  the  bacteriophage  is  absolute  when 
once  established,  and  is  negative  during  the  period  of  incubation. 
There  is  no  intermediary  state.  The  animals,  young  or  old, 
which  receive  the  test  inoculation  during  the  period  of  incubation 
die,  with  very  few  exceptions,  in  the  same  time  as  the  controls, 
even  if  this  inoculation  is  made  at  a  time  very  close  to  that  where 
all  the  immunized  animals  resist.  On  the  other  hand,  all  those 
which  are  tested  after  the  incubation  period  resist  without  pre- 
senting any  apparent  malaise,  whatever  the  test  dose  may  be. 
It  seems  indeed,  as  a  result  of  these  findings,  that  after  an  incuba- 
tion time,  more  or  less  protracted  according  to  the  amount  of 
bacteriophage  culture  injected,  a  period  during  which  the  animal 
remains  as  sensitive  as  a  normal  animal,  the  immunity  increases 
very  rapidly  once  its  manifestation  has  commenced.  In  a  word, 
the  release  of  immunity  is  abrupt. 

Effect  of  the  age  of  the  animals  on  the  acquisition  of  immunity 

We  have  seen  that  thirty-two  animals,  steers,  young  buffaloes, 
or  adult  buffaloes  of  less  than  twelve  years,  have  all  acquired 
an  immunity  that  approaches  the  refractory  condition  within 
twenty  hours  of  the  injection  of  0.25  cc.  of  the  bacteriophage  cul- 
ture. We  wished  to  see  how  this  would  compare  with  the  results 
obtained  in  old  animals. 

Three  buffaloes  between  fourteen  and  sixteen  years  and  five 
very  old  animals  no  longer  working  and  certainly  more  than 

4  Anaphylaxis  shows  a  phenomenon  of  the  same  order.  The  smaller 
the  sensitizing  dose,  the  shorter  the  period  of  time  before  the  animal 
is  sensitized.  For  example,  with  a  dose  of  0 . 001  cc.  of  serum  the  guinea  pig 
is  sensitized  after  about  fourteen  days,  with  5  cc.  sensitization  is  present 
only  after  several  months. 


254  THE   BACTERIOPHAGE 

twenty  years  old5  received  0.25  cc.  of  the  culture  of  bacteriophage. 
All  eight  were  tested  forty-three  days  later  by  the  inoculation  of 
1000  surely  fatal  doses  of  bacterium  barbone  culture  at  the  same 
time  as  a  normal  control  animal.  This  last  died  in  seventeen 
hours.  One  of  the  three  youngest  buffaloes  showed  no  reaction 
other  than  a  transitory  edema  at  the  site  of  the  inoculation,  the 
other  two  showed  a  voluminous  edema  and  were  obviously  sick, 
but  all  three  recovered  and  could  be  considered  normal  six  days 
after  the  test  inoculation.  The  five  very  old  buffaloes  suc- 
cumbed after  48,  53,  54,  60,  and  142  hours;  that  is,  after  a  time 
considerably  longer  than  the  control.  Fifteen  young  animals 
immunized  and  tested  at  the  same  time  failed  to  show  any  reac- 
tion to  the  test  injection. 

It  is  evident  that  although  the  test  dose  was  enormous,  that 
did  not  alter  the  fact  that  in  the  old  animals  the  acquisition  of 
immunity  was  much  more  difficult,  somewhat  in  proportion  to 
the  age.  The  relative  immunity  against  an  extremely  severe 
experimental  test  is  observed  only  in  these  old  animals;  with  the 
young  or  with  adults  in  the  prime  of  life,  the  immunity,  as  we 
have  seen,  is  absent  during  the  incubation  period  and  complete 
once  it  has  appeared  at  all. 

The  duration  of  the  immunity 

After  my  departure  from  Indo-China,  my  collaborator  M. 
Le  Louet,  continued  the  experiments  with  a  view  to  ascertaining 
the  duration  of  the  immunity  produced  by  the  inoculation  of  a 
culture  of  the  bacteriophage.  In  January,  1921,  he  injected  15 
steers,  aged  about  one  year,  with  a  cubic  centimeter  of  a  culture 
of  the  bacteriophage  that  was  about  one  month  old,  that  is,  a 
bouillon  culture  of  the  bacterium  of  barbone  which  had  been 

6  The  buffalo  usually  lives  about  twenty-five  or  thirty  years.  The 
Annamite  never  kills  a  buffalo;  old  and  no  longer  able  to  work,  it  is  fed  and 
cared  for  as  well  as  are  the  younger  animals.  The  attachment  of  the 
natives  for  these  buffaloes  is  such  that  it  is  difficult  to  find  a  person  who 
will  sell  one  of  these  animals.  Those  which  served  in  the  experiments  were 
procured,  some  through  the  agency  of  the  Governor  of  Cochin-China, 
M.  le  Gallen;  others  by  M.  Priv6,  Director  of  the  plantations  of  An  Loc  and 
Suzannah,  without  considering  the  possible  loss.  I  offer  them  my  sincere 
thanks. 


IMMUNIZATION   BY  MEANS   OF  THE  BACTERIOPHAGE  255 

lysed  by  the  bacteriophage  one  month  before  use.  In  March, 
1922,  all  of  the  animals  were  tested,  along  with  nine  controls, 
by  the  inoculation  of  0.1  cc.  of  a  virulent  culture  of  the  bacterium 
of  barbone.  The  virulence  of  this  culture  was  such  that  in 
amounts  of  0.002  cc.  it  regularly  killed  steers  in  less  than  thirty-six 
hours.  Of  the  animals  thus  infected  all  of  the  controls  died  in 
from  seventeen  to  twenty-three  hours  after  the  injection,  while  of 
the  vaccinated  animals  ten  resisted  without  any  evident  reaction 
and  five  died  in  from  two  to  five  days  after  inoculation. 

This  experiment  shows  that  fourteen  months  after  vaccination 
two-thirds  of  the  animals  possessed  an  immunity  sufficiently 
strong  to  enable  them  to  withstand  a  massive  dose  of  the  patho- 
genic bacterium. 

The  immunizing  principle 

Under  the  conditions  of  the  experiment,  that  is  to  say,  in  a 
non-contaminated  area,  what,  in  the  culture  of  the  bacteriophage, 
is  the  principle  which  brings  about  the  immunization? 

A  culture  of  the  bacteriophage  contains,  as  we  know: 

1.  The  bacteriophagous  ultramicrobes,  and 

2.  The  soluble  substances  contained  in  the  culture  medium. 
These  are  the   soluble  substances  derived  from  the  bacterial 
bodies  at  the  expense  of  which  the  bacteriophage  has  developed, 
the  lysins  secreted  by  these  ultramicrobes  and  which  remain  in 
the  medium  once  lysis  has  ended,  and  finally,  somewhat  later, 
the  anti-lysins  of  defense  secreted  by  the  bacteria. 

The  course  of  the  phenomenon  alone,  has  shown  us  already 
that  the  immunizing  principle  must  be  different  according  as 
the  immunity  is  developed  in  a  contaminated  area,  as  was  the 
case  in  the  experiments  made  on  typhosis,  or  in  a  non-contaminated 
area,  as  in  those  on  barbone.  In  the  first,  the  immunity  is 
acquired  immediately;  in  the  second,  it  becomes  effective  only 
after  an  incubation  period.  However,  direct  experiment  allows 
us  to  confirm  this  idea. 

1.  If  one  injects  steers,  by  the  subcutaneous  route,  with  from 
5  to  20  cc.  of  anti-barbone  bacteriophage  culture  it  is  possible 
to  isolate  the  active  ultramicrobe  from  the  tilood  throughout  the 
first  twenty-four  hours  after  the  injection.  After  this  period 


256  THE   BACTEKIOPHAGE 

they  have  disappeared.  Experiment  further  shows  that  the 
ultramicrobes  pass  quickly  into  the  intestine.  They  can  be 
isolated  from  the  intestine  within  about  twelve  hours  after  the 
injection  and  they  persist  there  for  a  somewhat  longer  time  than 
in  the  circulation:  for  two  or  three  days  (up  to  six  days  in  a  single 
case).  In  all  instances  they  have  disappeared  long  before  the 
immunity  is  established.  Let  us  repeat  that  this  applies  only 
to  the  case  where  the  introduction  of  the  bacteriophage  into  the 
organism  takes  place  in  a  territory  free  from  the  infection.  We 
have  seen,  for  example,  that  five  months  after  the  termination 
of  an  epizootic  of  barbone  it  is  still  possible  to  isolate  a  bacterio- 
phage active  for  the  pathogenic  bacterium  from  the  excreta  of 
buffaloes  which  have  resisted.  On  the  other  hand  experimenta- 
tion in  the  chicken  has  shown  us  that  the  activity  of  the  bacterio- 
phage for  the  pathogenic  bacillus  is  maintained  just  as  long  as 
the  experimental  animal  continues  to  ingest  these  bacteria. 

2.  Bablet  has  shown  that  the  bacteriophagous  germs  are  de- 
stroyed by  preservation  for  a  week  in  glycerine.  We  know  that 
this  substance  exerts  no  destructive  influence  on  either  the  dias- 
tases or  the  toxins.  It  may  be  assumed,  therefore,  that  in  a 
mixture  of  bacteriophage  culture  and  glycerine  the  ultramicrobes 
alone  will  be  destroyed  while  the  immunizing  substances  con- 
tained in  the  medium  will  remain  intact.  Starting  from  this 
hypothesis,  we  mixed  0.5  cc.  of  a  culture  of  the  anti-barbone 
bacteriophage  with  9.5  cc.  of  glycerine.  After  holding  the  mixture 
at  incubator  temperature  (37°C)  for  ten  days,  and  after  we  were 
assured  that  the  bacteriophagous  ultramicrobes  were  effectively 
destroyed,  we  inoculated  two  steers  with  this  liquid,  diluted  in 
500  cc.  of  saline.  Each  steer  received  then  0.25  cc.  of  the  original 
culture.  Tests  after  forty-five  days,  respectively  with  5  and  50 
fatal  doses  of  a  culture  of  the  bacterium  of  barbone,  showed  that 
these  two  animals  resisted.  They  had  acquired  an  immunity  in 
spite  of  the  destruction  of  the  bacteriophagous  ultramicrobes. 

In  the  case  of  experimental  barbone  the  tests  were  made  in  a 
barbone-free  region,  and  the  principle  which  is  responsible  for 
the  development  of  the  immunity  is  most  probably  constituted 
of  the  substance  of  the  bacterial  cells.  The  role  which  the  bac- 
teriophage plays  here  is  to  dissolve  the  bacteria,  in  which  condi- 


IMMUNIZATION   BY  MEANS   OF  THE  BACTERIOPHAGE  257 

tion  the  bacterial  substance  is  in  a  state  particularly  adapted  to 
stimulating  the  cells  of  the  body  which  enter  into  the  production 
of  organic  immunity.  The  substance  of  the  bacterial  body  dis- 
solves in  the  medium  under  the  influence  of  the  lysins  secreted 
by  the  ultramicrobes,  but  it  is  not  present  in  the  same  condition 
as  in  the  body  of  the  living  bacterium,  for  the  bacteriophage  does 
not  simply  produce  a  disintegration.  This  is  shown  by  the  fact 
that  the  culture  medium  becomes  perfectly  limpid,  whereas  the 
medium  remains  cloudy  when  a  simple  disintegration  takes  place. 
As  we  have  seen  in  several  tests,  the  destruction  of  the  bacterium 
by  the  activities  of  the  lysins — the  diastases — is  a  process  of 
solution.  Indeed,  it  is  rather  the  substances  composing  the 
bacterial  body  which  are  dissolved.  This  process  is  of  necessity 
accompanied  by  a  change  in  state.  It  is,  then,  not  proper  to 
speak  of  the  bacterial  substance  as  the  principle  which  provokes 
the  acquisition  of  immunity;  it  is  in  reality  the  products  result- 
ing from  the  degradation,  under  the  influences  of  lysins  secreted 
by  the  ultramicrobes,  of  the  substances  composing  the  bacterial 
cells  which  are  effective. 

It  is  obvious  that  this  is  yet  only  an  hypothesis,  experiment 
showing  only  that  the  principle  which  provokes  the  appearance 
of  immunity  is  not,  under  the  conditions  of  the  experiment,  the 
bacteriophage  considered  as  a  living  being.  Aside  from  the 
dissolved  bacterial  substance  do  the  diverse  principles  present 
in  the  culture,  namely,  the  bodies  of  dead  ultramicrobes,  the 
lysins,  and  eventually  the  anti-lysins,  play  any  part  in  the  pro- 
duction of  immunity?  In  the  present  state  of  these  investiga- 
tions it  is  impossible  to  affirm  or  deny  this. 

We  have  tested  the  action  of  temperature  on  the  immunizing 
element  contained  in  the  bacteriolysate.  To  this  end,  we  have 
repeated  the  experiment  of  the  culture  of  the  bacteriophage  treated 
with  glycerine,  with  the  difference  that  the  culture  has  previously 
been  subjected  to  a  temperature  of  56°C.  maintained  for  a  half 
hour.  Two  steers  have  each  received  a  dose  of  this  culture, 
heated  and  glycerinized,  corresponding  to  0.25  c.c.  of  the  original 
culture.  After  forty-five  days  they  were  tested,  the  one  with  five, 
the  other  with  fifty,  fatal  doses  of  barbone  culture.  The  first  re- 
sisted, the  second  died.  The  immunizing  principle  contained  in  the 


258  THE   BACTERIOPHAGE 

bacteriophage  culture  is  not  destroyed  but  is  sensibly  weakened  by 
heating  for  a  half  hour  at  56°C. 

Although  it  is  not  yet  possible  to  know  with  certainty  the 
nature  of  the  process  which  controls  the  development  of  organic 
immunity,  we  are  at  least  able  to  recognize  the  result  and  to  note 
the  property  which  distinguishes  the  animal  immunized  by  an 
injection  of  the  bacteriophage  from  a  normal  animal. 

In  the  case  of  the  immunity  acquired  as  a  result  of  an  attack 
of  a  contagious  disease  the  blood  possesses  preventive  properties. 
The  blood  of  immunized  animals  enjoys  the  same  property,  as 
the  following  experiments  show. 

I.  Steer  no.  54  received  on  November  5,  0.25  cc.  of  an  anti-barbone  bac- 
teriophage culture.    Fourteen  days  later  500  cc.  of  blood  was  taken  into 
a  flask  containing  25  cc.  of  a  10  per  cent  solution  of  sodium  citrate.     The 
blood  was  immediately  injected  into  the  jugular  vein  of  steer  no.  43.     This 
last  animal  was  tested  twenty-three  hours  later  by  the  injection  of  1000 
fatal  doses  of  the  bacterium  of  barbone  culture.     It  failed  to  show  the 
least  evidence  of  infection.    A  control  died  in  twenty-three  hours.    Steer 
no.  54  likewise  resisted  the  inoculation  of  1000  fatal  doses,  given  on  Decem- 
ber 1st. 

II.  The  experiment  given  above  was  repeated.    Steer  no.  112  received 
into  the  jugular  vein  500  cc.  of  blood  from  steer  no.  95.     Both  of  them 
resisted  the  test  injections. 

III.  Steer  no.  104  received  on  December  29  a  subcutaneous  injection 
of  0.04  cc.  of  a  culture  of  the  bacteriophage.     Four  days  later  500  cc. 
of  blood  were  taken,  as  before,  and  this  was  transfused  into  steer  no.  108. 
The  next  day  the  two  steers  resisted  the  inoculation  of  five  fatal  doses, 
which  killed  the  control  animal  in  thirty-two  hours. 

IV.  The  above  experiment  (III)  was  repeated.     The  steer  which  received 
the  blood  of  the  immunized  animal  was  not  tested  by  the  inoculation  of  50 
fatal  doses  until  forty-five  days  after  the  transfusion.     It  resisted,  without 
showing  any  apparent  disturbance,  as  did  also  the  steer  which  was  immu- 
nized directly. 

This  last  experiment  does  not,  however,  prove  anything  with 
regard  to  the  duration  of  passive  immunity  conferred  by  the  blood 
of  an  immunized  animal,  for  it  was  performed  with  homologous 
blood,  and  we  know  that  an  immunity  thus  produced  is  of  much 
longer  duration  than  when  produced  with  heterologous  blood. 
In  all  cases,  the  immunity  thus  conferred  is  extremely  powerful 
and  these  experiments  open  the  way  for  further  investigations 


IMMUNIZATION   BY  MEANS  OF  THE  BACTERIOPHAGE  259 

on  the  production  of  therapeutic  sera  in  animals  immunized  by  a 
single  injection  of  an  active  bacteriophage,  not  only  for  barbone, 
but  for  other  diseases  as  well. 

One  might  conceive  that  the  "principle"  which  is  contained 
in  the  blood  of  the  immunized  animal  and  which  confers  the  pas- 
sive immunity  does  not  differ  from  the  culture  of  the  bacteriophage 
which  persists  for  a  certain  length  of  time  in  the  circulation. 
But  this  is  impossible,  for  if  the  blood  is  taken  at  a  time  sufficiently 
close  to  the  immunizing  injection  of  the  bacteriophage  it  is  in 
no  way  effective.  That  is,  blood  taken  during  the  incubation 
period  confers  no  immunity  to  the  transfused  animal. 

Steers  nos.  89  and  90  received  on  December  19,  0.25  cc.  of  the  bacte- 
riophage culture  subcutaneously.  Sixteen  days  later  500  cc.  of  blood  were 
withdrawn  from  each  animal  and  transfused  into  steers  nos.  92  and  93. 
The  four  animals,  tested  the  next  day,  died  with  no  greater  delay  than  the 
controls.  Steer  no.  46  received  on  November  5,  20  cc.  of  the  culture  of 
bacteriophage.  On  November  19,  500  cc.  of  blood  were  taken  and  trans- 
fused into  steer  no.  42.  These  two  animals  died  in  the  same  time  as  the 
control  after  a  test  injection. 

As  is  to  be  seen,  the  incubation  period  of  immunity  in  animals 
which  receive  the  immunizing  injection  of  bacteriophage  culture 
parallels  the  appearance  of  the  protective  power  in  their  blood. 
Immunity  develops  abruptly;  in  the  same  way  the  protective 
power  of  the  blood  manifests  itself  suddenly,  and  at  the  same 
moment. 

What  then,  is  the  immunizing  principle  which  makes  its  sudden 
appearance  in  the  blood  at  the  moment  when  immunity  is  es- 
tablished, even  in  animals  which  have  received  only  the  minimal 
dose  of  a  single  drop  of  the  culture  of  the  bacteriophage?  Can 
it  be  an  amboceptor?  By  no  means,  for  the  complement  fixa- 
tion reaction  shows  that  the  sera  of  animals  immunized  with 
cultures  of  the  bacteriophage  do  not  contain  a  specific  ambo- 
ceptor in  detectable  quantity.  In  conducting  the  reaction  of 
Bordet  with  even  0.5  cc.  one  obtains  an  exactly  comparable  he- 
molysis,  of  the  same  intensity  and  in  the  same  time,  as  that  which 
occurs  in  a  control  tube  containing  the  same  quantity  of  normal 
serum. 


260  THE   BACTEEIOPHAGE 

Examination  of  the  opsonic  power  of  two  of  these  sera  gave 
indices  of  0.3  and  0.4;  indices  which  are  essentially  negative.6 

The  serum  containing  the  protective  principle  does  not  con- 
tain inhibiting  substances  or  even  substances  delaying  the  growth 
of  the  bacterium  of  barbone.  Bouillon,  mixed  with  such  a  serum, 
in  any  proportion  (from  0.05  cc.  to  3  cc.  per  10  cc.  of  bouillon), 
with  or  without  the  addition  of  fresh  guinea  pig  serum,  furnishes 
a  medium  which,  when  inoculated,  gives  luxuriant  cultures  of  the 
bacterium  of  barbone. 

Finally,  the  serum  contains  no  traces  of  agglutinins. 

Organic  immunity,  then,  is  not  due  to  the  presence  of  an  am- 
boceptor,  nor  to  the  presence  of  an  opsonin  in  the  blood  of  the 
vaccinated  subjects.  The  blood  contains  neither  agglutinins 
nor  inhibiting  substances.  The  immunity  is  most  probably 
antitoxic. 

We  have  seen  in  the  experiments  performed  on  avian  typhosis, 
that,  in  an  infected  area,  the  protection  of  the  animal  is  immediate 
and  that  this  protection  is  assured  only  by  the  presence  of  bac- 
teriophagous  ultramicrobes  virulent  for  the  pathogenic  bac- 
terium. We  have  again  found  this  immediate  immunity  in  the 
case  of  barbone.  It  is  that  which  protected  steers  nos.  103  and 
107  against  the  inoculation  of  five  fatal  doses  of  culture  when 
given  only  twenty-four  hours  after  the  injection  of  the 
bacteriophage. 

In  typhosis,  this  heterologous  immunity  has  been  permanent, 
for  the  daily  reinfections  which  occur  in  the  infected  area  allow 
the  bacteriophage  to  multiply  at  the  expense  of  the  pathogenic 
bacteria  ingested  and  thus  to  maintain  its  virulence  for  this 
bacterium.  In  barbone,  this  same  thing  takes  place  in  an  in- 
fected area,  since  we  have  seen  that  the  bacteriophage  virulent 
for  the  bacterium  of  barbone  was  present  in  the  intestine  of 
buffaloes  five  months  after  the  complete  disappearance  of  the 
epizootic. 

6  We  have  seen  in  Part  I  that  the  lysin  secreted  by  the  bacteriophage 
possesses  a  very  high  opsonic  power.  The  organism  must  respond  to  an 
injection  of  lysin  (certainly  present  in  the  culture  of  the  bacteriophage) 
by  the  production  of  an  anti-opsonic  antibody. 


IMMUNIZATION   BY  MEANS  OF  THE  BACTERIOPHAGE  261 

In  a  non-infected  region,  and  this  was  the  case  in  the  experi- 
ments performed  on  barbone,  the  mechanism  is  not  the  same. 
In  the  absence  of  reinfection  the  bacteriophage  active  for  the 
bacterium  is  eliminated  very  rapidly  from  the  organism,  since  it 
is  not  able  to  multiply  at  the  expense  of  this  bacterium.  The 
heterologous  immunity  disappears  with  it, — that  is  to  say,  after 
one  or  two  days, — and  the  animal  then  becomes  susceptible.  It 
remains  in  this  condition  throughout  the  entire  duration  of  the 
incubation  of  the  organic  immunity,  which  develops  under  the 
influence  of  the  soluble  products  contained  in  the  culture  of  the 
bacteriophage.  Once  this  organic  immunity  is  established  the 
animal  is  refractory. 

We  will  see  in  connection  with  dysentery,  that  when  a  culture 
of  the  bacteriophage  is  injected  the  organism  responds  by  the 
production  of  an  antitoxin.  It  is  probable  that  the  same  thing 
takes  place  in  barbone  and  that  the  protective  principle  present 
in  the  blood,  since  it  is  neither  an  amboceptor  nor  an  opsonin, 
is  likewise  an  antitoxin ;  the  response  of  the  organism  to  the  in- 
jection of  the  modified  substance  of  the  lysed  bacterial  cells 
contained  in  the  culture  of  the  bacteriophage. 

To  summarize:  the  injection  of  the  buffalo  or  of  cattle,  with 
a  culture  of  bacteriophage  active  for  the  bacterium  of  barbone 
confers  : 

1.  An  heterologous  immunity,  solely  due  to  the  presence  in 
the  body  of  bacteriophagous  ultramicrobes  virulent  for  the  bac- 
terium of  barbone,  which  assures  the  destruction  of  the  bacteria 
upon   their   introduction    into   the    organism.     This   immunity 
terminates  just  as  soon  as  the  ultramicrobes  are  eliminated  from 
the  body.     In  the  absence  of  frequent  reinfections  this  elimina- 
tion is  very  rapid,  since  the  continued  growth  and  the  mainte- 
nance of  virulence  can  not  persist. 

2.  An  homologous  immunity,  or  organic  and  powerful  immunity, 
induced  by  a  reaction  of  the  organism  of  the  animal  to  the  soluble 
principles  contained  in  the  culture  of  bacteriophage  injected. 
This  organic  immunity  is  characterized  principally  by  the  appear- 
ance in  the  blood  of  an  extremely  potent  immunizing  substance — 
probably  an  antitoxin.     The  organic  immunity  establishes  itself 
abruptly  after  an  incubation  period,  which  varies  with  the  dose  in- 
jected, being  longer  as  the  amount  of  injected  culture  is  increased. 


262  THE   BACTERIOPHAGE 

A  single  injection  of  0.04  cc.,  or  less  than  a  normal  sized  drop, 
into  a  steer  of  100  kgms.  weight  places  the  animal  within  4 
days  in  a  condition  where  it  can  withstand  a  test  inoculation 
of  five  fatal  doses.  Sixty  days  later  the  animal  resists  a 
test  inoculation  representing  fifty  surely  fatal  doses. 

The  blood  of  an  immunized  animal  injected  into  a  normal 
animal  confers  on  the  latter  a  passive  immunity  as  solid  as 
that  enjoyed  by  the  actively  immunized  one  itself,  even  if  this 
last  one  has  received  but  a  single  injection  of  0.04  cc.  of  culture 
of  the  bacteriophage.  And  this  passive  immunity,  under  ex- 
perimental conditions  at  least,  is  still  intact  forty-five  days  after 
the  injection  of  the  blood. 

IMMUNIZATION   AGAINST    DYSENTERY 

"The  cultures  of  Shiga  lysed  by  the  invisible  microbe,  which 
are  in  reality  cultures  of  the  anti-microbe,  possess  the  property 
ctf  immunizing  the  rabbit  against  a  dose  of  Shiga  bacilli  which 
will  kill  the  controls  in  five  days."  This  statement  is  taken  from 
my  first  communication  on  the  bacteriophage.  The  experi- 
mental data  upon  which  this  affirmation  was  based  are  given  in 
the  following  protocols. 

The  rabbit,  although  naturally  refractory  to  bacillary  dysentery 
is,  on  the  contrary,  susceptible  to  the  inoculation  of  dysentery 
toxin.  This  animal  could,  then,  be  utilized  for  the  preliminary 
antitoxic  immunization  experiments.  The  following  experiments 
showed  at  first  that  the  culture  of  anti-Shiga  bacteriophage,  a 
short  time  after  lysis,  is  toxic,  although  to  a  less  degree  than  is  a 
normal  culture  of  Shiga  bacilli. 

Rabbit  no.  1.  One  cubic  centimeter  of  a  normal  culture  of  Shiga  bacilli 
was  injected  intravenously  on  August  10.  The  animal  died  on  August  16. 

Rabbit  no.  2.  Two  cubic  centimeters  of  a  normal  culture  of  Shiga 
bacilli  were  injected  subcutaneously  on  August  10.  The  animal  died 
on  August  16. 

Rabbit  no.  3.  One  cubic  centimeter  of  a  Shiga  bacillus  culture  which  had 
been  subjected  to  lysis  for  six  hours  was  injected  intravenously.  (The 
amount  of  bacillary  substance  here  was  the  same  as  in  the  preceding.) 
The  rabbit  lived. 

Rabbit  no.  4.  Two  cubic  centimeters  of  a  Shiga  culture  which  had  been 
lysed  for  six  hours  were  injected  subcutaneously.  The  animal  died  on 
August  16.  This  rabbit  had  also  been  injected  on  August  10. 


IMMUNIZATION  BY  MEANS   OF  THE  BACTERIOPHAGE  263 

Six  days  after  the  completion  of  the  lysis  the  toxicity  of  the 
culture  was  markedly  diminished,  as  the  following  tests  show: 

Rabbit  no.  5.  Two  cubic  centimeters  of  a  Shiga  bacillus  culture  which 
had  been  lysed  for  six  days  were  injected  intravenously  on  August  10. 
The  animal  lived. 

Rabbit  no.  6.  Three  cubic  centimeters  of  the  Shiga  culture  which  had 
been  lysed  for  six  days  were  injected  subcutaneously  on  August  10.  This 
rabbit  also  lived. 

Rabbit  no.  7.  Five  cubic  centimeters  of  the  Shiga  culture  which  had 
been  lysed  for  six  days  were  injected  intravenously  on  August  10.  The 
rabbit  died  on  August  21. 

When  the  tests  were  done  with  a  Shiga  culture  which  had  been 
lysed  for  a  month  the  toxicity  had  disappeared,  as  is  shown  by 
the  following. 

Rabbit  no.  8.  Fifteen  cubic  centimeters  of  Shiga  culture  which  had  been 
lysed  for  one  month  were  injected  subcutaneously.  The  rabbit  lived. 
The  injection  was  given  on  August  10. 

Rabbit  no.  9.  Ten  cubic  centimeters  of  this  same  culture  were  injected 
intravenously  on  August  10.  This  rabbit  lived. 

The  following  protocol  illustrates  an  immunization  experiment. 

On  August  23,  eight  rabbits  received  a  subcutaneous  injection  of  0.25 
cc.  of  a  culture  of  the  anti-Shiga  bacteriophage,  two  months  after  the  lysis 
was  completed.  These  animals  were  tested  by  the  injection  of  3  cc.  of  a 
twenty-four  hour  bouillon  culture  of  Shiga  bacilli.  For  the  strain 
employed  this  represented  two  surely  fatal  doses.  The  strain  of  Shiga 
used  in  the  test  differed  from  that  used  to  prepare  the  suspension  lysed  by 
the  bacteriophage. 

Rabbit  no.  10;  tested  after  twenty-eight  hours.     Died  six  days  later. 

Rabbit  no.  11 ;  tested  after  four  days.    Died  five  days  later. 

Rabbit  no.  12;  tested  after  six  days.     Lived. 

Rabbit  no.  13;  tested  after  eight  days.     Lived. 

Rabbit  no.  14;  tested  after  ten  days.     Lived. 

Rabbit  no.  15;  tested  after  one  month.     Lived. 

Rabbit  no.  16;  tested  after  two  months.    Lived. 

Rabbit  no.  17;  tested  after  three  months.     Lived. 

All  the  control  rabbits  inoculated  with  half  the  dose,  that  is,  with  1.5 
cc.  of  the  Shiga  culture,  died  in  from  four  to  seven  days. 

The  rabbit  is  therefore,  immunized  against  two  surely  fatal 
doses  of  B.  dysenteriae  Shiga  culture  by  the  injection  of  a  quarter 


264  THE   BACTERIOPHAGE 

of  a  cubic  centimeter  of  a  culture  of  the  anti-Shiga  bacteriophage. 
The  antitoxic  immunity  is  established  six  days  after  the  injec- 
tion and  persists  for  at  least  three  months. 

In  an  experiment  of  this  kind  there  can  be  no  question  of  the 
nature  of  the  process.  The  bacteriophage  as  a  living  being  can 
not  be  the  cause  of  the  immunity.  The  responsible  agent  must 
be  the  soluble  principles  contained  in  the  culture  medium.7 

Before  undertaking  experiments  on  man  I  had  to  assure  myself 
that  the  administration  of  cultures  of  the  anti-Shiga  bacterio- 
phage caused  no  reaction.  First,  I  ingested  increasing  quantities 
of  the  cultures,  aged  from  six  days  to  a  month,  from  one  to  thirty 
cubic  centimeters,  without  detecting  the  slightest  malaise.  Three 
persons  in  my  family  next  ingested  variable  quantities  several 
times  without  showing  the  least  disturbance.  I  then  injected 
myself  subcutaneously  with  one  cubic  centimeter  of  a  forty-day 
old  culture.  There  was  neither  a  local  nor  a  general  reaction. 
In  all  the  cases,  twenty-four  hours  after  the  ingestion  or  after 
the  injection,  I  was  able  to  isolate  from  the  stools  a  bacteriophage 
possessing  for  the  Shiga  bacillus  an  activity  equal  to  that  of  the 
ultramicrobe  administered.  More  recently  G.  Eliava  has  re- 
ceived by  subcutaneous  injection  5  cc.  of  a  culture  of  the  anti- 
Shiga  bacteriophage  aged  thirty  days.  No  reaction,  local  or 
general,  followed. 

It  is  known  that  the  subcutaneous  injection  of  Shiga  bacilli, 
killed  by  any  procedure  whatsoever,  can  not  be  performed  be- 
cause of  the  extremely  violent  reaction^  produced,  and  which 
are  due  to  the  toxicity  of  the  germ.  This  is  precisely  the  reason 
that  vaccine  prophylaxis  is  not  applied  to  dysentery  as  it  is  in 
the  case  of  typhoid.  The  absolute  innocuity  of  injections  of 

7  Several  immunizing  experiments  with  the  bacteriophage  for  B.  typhosus 
and  for  the  paratyphoid  organisms  have  been  performed  upon  laboratory 
animals,  both  rabbits  and  guinea  pigs.  In  all  cases  these  showed  a  perfect 
immunization; — provided  it  is  permissible  to  employ  the  word  immuniza- 
tion when  the  process  is  carried  out  in  refractory  animals. 

Not  attributing  any  value  to  experiments  of  this  type  I  have  not  included 
them  in  the  monograph.  In  all  cases  the  bacteriophage  administered, 
either  when  given  by  subcutaneous  injection  or  by  the  buccal  route,  has 
been  isolated  a  few  hours  later  from  the  intestinal  tract. 


IMMUNIZATION   BY  MEANS   OF  THE   BACTERIOPHAGE  265 

the  anti-Shiga  bacteriophage  cultures,  which  contain  the  sub- 
stance of  the  bacterial  bodies  in  a  dissolved  state,  shows  indeed 
that  these  substances  undergo  profound  modifications  under  the 
influence  of  the  lysins  secreted  by  the  bacteriophagous  ultrami- 
crobe.  Nevertheless,  these  new  substances  possess  a  specific 
immunizing  power  much  more  potent  than  the  original  substance. 
The  experiments  on  rabbits,  and  in  particular  the  results  secured 
in  immunization  against  barbone,  demonstrate  this  beyond  pos- 
sible doubt. 

Prophylactic  vaccination  against  bacillary  dysentery  by  means 
of  cultures  of  the  anti-dysentery  bacteriophage  is  therefore 
applicable  to  man.  In  practice,  quite  naturally,  the  prophylactic 
injections  should  be  performed  with  a  mixture  of  bacteriophage 
cultures — anti-Shiga,  anti-Flexner,  and  anti-Hiss.  Such  a  mix- 
ture would  constitute  a  polyvalent  dysentery  vaccine. 

The  Shiga  bacillus  is  one  of  the  most  toxic  organisms  known, 
and  it  may  be  assumed  that  the  harmlessness  of  injections  of 
such  a  culture  indicates  a  general  law,  whatever  may  be  the 
bacterium  against  which  the  bacteriophage  culture  is  prepared. 
In  order  to  test  this  hypothesis,  I  injected  myself,  subcutaneously, 
with  half  a  cubic  centimeter  of  anti-plague  bacteriophage.  No  reac- 
tion, either  general  or  local,  followed.  Stool  examination  made 
twenty-four  hours  after  the  injection  showed  that  the  bacterio- 
phage, equal  in  virulence  to  that  injected,  was  present.  The 
inoculation  experiment  was  repeated  with  anti-typhoid  bac- 
teriophage. G.  Eliava  repeated  it  with  the  anti-staphylococcus 
bacteriophage,  and  the  same  results  were  secured  in  both  cases. 
These  observations  are  confirmed  in  part  by  another  fact,  ob- 
served in  several  tests,  that  following  the  administration  of  the 
bacteriophage,  either  by  injection  or  by  ingestion,  the  bacterio- 
phage passes  in  a  short  time  into  the  intestine.  It  is  eliminated 
rapidly  if  it  fails  to  encounter  the  bacterium  against  which  it 
has  a  virulence,  that  is  to  say,  in  an  uninfected  individual. 
On  the  contrary,  it  grows  and  maintains  its  virulence  if  it  is  in 
contact  with  this  bacterium,  a  condition  which,  as  we  have  seen 
in  several  instances,  is  produced  in  an  infected  environment 
among  animals  which  remained  healthy,  or  which  had  been  in- 
fected and  were  recovered. 


266  THE   BACTERIOPHAGE 

After  being  assured  of  the  innocuity  of  the  ingestion  of  cultures 
of  the  anti-Shiga  bacteriophage,  this  treatment  was  applied  for 
therapeutic  purposes  to  patients  affected  with  bacillary  dysentery.8 
As  in  the  experimental  work,  so  also  here  in  the  clinical  tests, 
the  therapy  has  been  limited  to  those  cases  in  which  the  etiology 
of  the  infection  was  proved  by  the  isolation  of  the  pathogenic 
organism,  and  where,  in  addition,  the  virulence  of  the  intestinal 
bacteriophage  was  negative  toward  the  different  dysentery  bacilli 
at  the  time  of  the  administration  of  the  culture  of  bacteriophage. 
It  is  evident  that  in  routine  practice  it  would  not  be  necessary  to 
investigate  all  these  points,  especially  since  the  administration 
of  the  bacteriophage  cultures  is  always  inoffensive. 

In  each  of  the  following  cases  the  only  treatment  instituted 
has  been  the  ingestion  of  the  culture  of  the  bacteriophage. 

Robert  K.  .  .  .  (eleven  years).  This  is  a  case  of  bacillary  dysentery 
of  moderate  severity  with  from  5  to  7  bloody  stools  a  day. 

August  1.  The  stool  examination  showed:  B.  dysenteriae  Shiga  present. 

The  intestinal  bacteriophage  with  virulences  as  follows:  B.  coli  ++, 
Shiga  0,  Flexner  0.  Hiss  0. 

August  2.  At  10  o'clock  in  the  morning  the  patient  ingested  2  cc.  of  a 
anti-Shiga  bacteriophage  culture.  This  culture  had  been  lysed  for  thirty- 
five  days.  During  the  afternoon  of  this  day  there  were  3  bloody  stools,  in 
the  evening  there  was  one  stool  and  that  was  free  of  blood. 

August  3.  During  this  day  there  was  only  the  one  formed  stool.  Exami- 
nation showed:  B.  dysenteriae  Shiga  absent. 

The  intestinal  bacteriophage  with  virulences  as  follows:  B.  coli  +  +  +  +  , 
Shiga  ++++,  Flexner  +  ++,  Hiss  +++. 

8  These  experiments  have  been  made  with  the  assistance  of  M.  Nadal, 
on  the  service  of  Pr.  Hutinel,  at  the  Hopital  des  Enfants  Malades. 

Tests  have  also  been  made  in  cases  of  toxic  diarrhea  of  infants,  but  they 
will  not  be  discussed  here  since  a  conclusion  regarding  them  has  not  yet 
been  reached.  In  those  cases  there  is  an  especial  difficulty,  for  the  path- 
ogenic organism  is  still  unknown.  It  was  at  first  thought  that  this  might 
be  determined  through  the  ability  to  isolate  and  cultivate  an  active  strain 
of  bacteriophage  which  might  be  used  for  curative  purposes.  It  is  indeed 
probable  that  there  is,  not  one,  but  several  diarrheas  of  infants  caused  by 
different  bacterial  types,  as  the  experiments  of  Nobe"court  made  during 
the  past  few  years  would  also  indicate.  The  solution  of  the  problem  is  not 
impossible  but  it  would  be  necessary  to  administer  to  the  affected  infants 
a  mixture  of  cultures  of  diverse  strains  of  the  bacteriophage,  active  against 
the  diverse  bacterial  types  capable  of  inciting  the  diarrhea.  It  can  readily 
be  conceived  that  under  such  circumstances  the  investigation  must  be 
protracted. 


IMMUNIZATION   BY  MEANS   OF  THE  BACTERIOPHAGE  267 

August  8.  The  intestinal  bacteriophage  was  active  as  follows:  B.  coli 
-f ++,  Shiga  +,  Flexner  0,  Hiss  +. 

August  9.    The  patient  was  discharged  from  the  hospital. 

Andre"  B.  .  .  .  (ten  years).  A  case  of  bacillary  dysentery  of  moderate 
severity.  During  the  period  from  August  25  to  29  inclusive  there  were  9 
to  11  bloody  stools  a  day. 

August  28.     Stool  examination  showed:  B.  dysenteriae  Shiga  present. 

Intestinal  bacteriophage  active  as  follows:  B.  coli  +,  Shiga  0,  Flexner 
0,  Hiss  0. 

August  29.  At  4  p.m.  the  patient  ingested  2  cc.  of  an  anti-Shiga  bac- 
teriophage culture.  The  culture  had  been  lysed  for  two  months. 

August  30.  There  was  one  bloody  stool  in  the  morning  and  during 
the  afternoon  and  the  night  there  were  5  stools,  none  of  which  showed 
any  blood. 

August  31.    There  were  3  fluid,  but  not  bloody,  stools. 

Examination  showed:  Shiga  bacilli  not  present. 

The  intestinal  bacteriophage  active  as  follows:  B.  coli  +  +  ++,  Flexner 
++,  Hiss  +++,  Shiga  +  +  +  +  . 

September  1.     There  was  one  fluid  stool,  without  blood. 

September  2.     There  was  one  fluid  stool,  without  blood. 

September  3.  There  was  one  formed  stool.  Examination  of  the  intes- 
tinal bacteriophage  showed:  B.  coli  +++,  Shiga  ++++,  Flexner  +  ++, 
Hiss  +. 

September  8.  Reactions  with  the  intestinal  bacteriophage  were: 
B.  coli  ++,  Shiga  0,  Flexner  0,  Hiss  +. 

September  9.    The  patient  was  discharged  from  the  hospital. 

Robert  D.  .  .  .  (twelve  years).  This  patient  had  a  very  severe  dysen- 
tery, with  vomiting,  cold  sweats,  chilling  of  the  extremities,  and  involun- 
tary and  uncountable  stools. 

September  8.  The  stools  could  not  be  counted.  They  were  fetid, 
purulent,  and  streaked  with  blood.  Examination  showed:  B.  dysenteriae. 
Shiga  present;  about  1  out  of  every  10  colonies  on  the  plates  was  the  dysen- 
tery bacillus. 

The  intestinal  bacteriophage  showed  no  virulence  for  B.  coli,  or  for  the 
Shiga,  Flexner  or  Hiss  organisms. 

September  9.  Two  cubic  centimeters  of  an  anti-Shiga  bacteriophage 
culture  were  ingested  at  11  o'clock.  This  culture  had  been  lysed  for  three 
and  one-half  months.  During  the  afternoon  and  the  night  the  stools 
became  less  numerous  but  continued  bloody. 

September  10.  There  were  6  fluid  stools,  without  blood.  Examination 
showed:  B.  dysenteriae  Shiga,  not  present. 

Intestinal  bacteriophage  active  as  follows:  B.  coli  +  +++,  Shiga 
+  +  ++,  Flexner  ++++,  Hiss  +  +  +  . 

September  11.    There  were  2  normal,  formed  stools. 

September  20.     The  patient  was  discharged  from  the  hospital. 


268  THE   BACTERIOPHAGE 

Julien  D.  .  .  .  (three  and  one-half  years).  This  was  a  case  of  very 
severe  dysentery.  The  general  condition  of  the  patient  was  very  bad. 
A  sister  of  the  patient  had  died  at  home  of  dysentery  on  September  8. 

From  the  llth  to  the  13th  of  September  the  number  of  stools,  all  of  which 
were  bloody,  could  not  be  counted. 

September  13.  The  patient  entered  the  hospital.  Examination  showed  : 
B.  dysenleriae  Shiga  present,  the  dysentery  bacilli  constituting  about  4 
of  every  5  colonies  on  the  plates. 

The  intestinal  bacteriophage  was  without  activity  for  either  B.  coli 
or  the  dysentery  organisms. 

September  13.  The  patient  ingested  2  cc.  of  anti-Shiga  bacteriophage 
culture  at  5  o'clock.  This  culture  had  been  lysed  for  fifteen  days. 

September  14.  There  were  6  bloody  stools.  The  intestinal  bacterio- 
phage showed  virulences  as  follows:  B.  coli  +++,  Shiga  +++,  Flexner 
++,  Hiss  +. 

September  15.  During  the  day  there  was  one  bloody  stool.  There  were 
also  5  stools  without  blood.  Examination  showed:  B.  dysenteriae  Shiga 
absent. 

The  intestinal  bacteriophage  with  activities  as  follows:  B.  coli  +  +++, 
Shiga  +  +++,  Flexner  +  ++,  Hiss  +++. 

September  16.  There  were  4  stools,  all  without  blood.  The  intestinal 
bacteriophage  showed:  B.  coli  ++++,  Shiga  ++++,  Flexner  ++,  Hiss 


September  17.  During  the  day  there  were  one  fluid  stool  and  2  formed 
stools.  The  intestinal  bacteriophage  showed:  B.  coli  +-  f-++,  Shiga 
++++,  Flexner  ++,  Hiss  +. 

September  18.  There  were  2  formed  stools.  The  intestinal  bacterio- 
phage was  virulent  as  follows:  B.  coli  ++++,  Shiga  ++++,  Flexner  +, 
Hiss  ++. 

September  26.  The  patient  was  discharged  from  the  hospital.  On 
this  date  the  virulence  of  the  intestinal  bacteriophage  was:  B.  coli  +++» 
Shiga  0,  Flexner  0,  Hiss-f. 

Emile  D.  .  .  .  (seven  and  one-half  years).  This  patient  was  a  brother 
of  the  preceding  case,  and  showed  a  very  severe  dysentery.  On  the  llth 
and  12th  of  September  there  were  20  to  25  fetid  stools,  fluid  but  not  bloody. 

September  12.  Examination  showed  that  the  Shiga  bacilli  were  very 
abundant.  The  intestinal  bacteriophage  was  inactive  for  B.  coli  or  for 
the  dysentery  organisms. 

September  13.  There  were  25  bloody  stools.  At  5  o'clock  the  patient 
ingested  2  cc.  of  bacteriophage  culture.  The  culture  had  been  lysed  for 
six  and  one-half  months. 

September  14.  There  were  4  bloody  stools  in  the  morning  and  2  stools 
without  blood  in  the  afternoon.  Examination  of  one  of  the  latter  showed 
no  Shiga  bacilli.  The  virulences  of  the  intestinal  bacteriophage  were: 
B.  coli  ++++,  Shiga  ++++,  Flexner  ++,  Hiss  +  ++. 


IMMUNIZATION   BY  MEANS   OF  THE  BACTERIOPHAGE  269 

September  15.     There  were  5  stools,  all  free  of  blood. 
September  16,  17,  and  18.    During  these  days  there  were  3  or  4  stools  a 
day.    None  of  these  contained  blood. 

September  19.    There  were  2  formed  stools  on  this  day. 
September  28.    The  patient  was  discharged  from  the  hospital. 

In  all  of  these  cases  the  general  condition  of  the  patient  has 
always  coincided  with  the  severity  of  the  intestinal  symptoms. 

Two  other  cases  of  dysentery  due  to  the  Shiga  bacillus,  treated 
in  the  same  manner,  but  outside  of  the  hospital,  gave  comparable 
results.  In  these  there  was  a  cessation  of  the  bloody  stools  with 
improvement  in  the  general  condition  in  the  twenty-four  hours 
immediately  following  the  administration  of  the  culture  of  anti- 
Shiga  bacteriophage. 

Obviously,  seven  cases  are  not  sufficient  to  afford  an  absolute 
proof  in  favor  of  the  specific  therapy  of  bacillary  dysentery  by 
means  of  bacteriophage  cultures.  However,  they  do  suffice  to 
show  that  the  ingestion  of  cultures  of  a  virulent  bacteriophage — 
virulent  for  the  infecting  bacillus — is  as  harmless  for  the  sick 
as  for  the  healthy  person.  They  also  show  that  the  ingested 
bacteriophage  traverses  the  upper  digestive  tract  in  man  as  it 
does  in  animals,  and  within  a  few  hours  will  be  found  in  the 
intestine  where  it  grows  at  the  expense  of  the  bacterium  for  which 
it  is  active.  Moreover,  these  seven  cases  acquire  a  significance 
from  the  fact  that  the  cultures  of  bacteriophage  restrained  the 
disease,  as  was  the  case  in  avian  typhosis,  as  is  shown  in  the 
results  of  the  experiments  which  have  been  recorded  and  in 
experiments  bearing  on  about  one  hundred  cases. 

All  these  facts  authorize  clinicians  to  continue  the  experimental 
treatment  on  a  more  elaborate  scale,  not  only  in  bacillary  dys- 
entery but  in  other  infectious  human  diseases  for  which  strains 
of  the  bacteriophage  have  been  isolated — typhoid  fever,  the 
paratyphoid  infections,  and  bubonic  plague.9 

Whatever  may  be  the  nature  of  the  disease  under  considera- 
tion, the  isolation  of  a  strain  of  the  bacteriophage  active  for  the 
pathogenic  bacterium  is  easy  once  it  appears  in  an  acute  disease 

9  It  may  also  be  suggested  that  the  bacteriophage  may  have  an  applica- 
tion in  surgery,  as  in  the  treatment  of  wounds  or  in  peritonitis,  either  as 
a  preventive  when  an  infection  is  to  be  feared,  or  as  a  therapeutic  agent 
when  infection  has  once  appeared. 


270  THE   BACTERIOPHAGE 

where  the  bacterium  is  known  and  cultivable.  It  is  only  neces- 
sary to  apply  the  principles  which  have  been  discussed  in  the 
course  of  this  work.10 

10  Since  the  publication  of  the  French  edition  of  this  work  Bruynoghe 
and  Maisin  at  the  Bacteriologic  Institute  of  Louvain,  and  Beckerich  and 
Hauduroy  at  the  Institute  of  Hygiene  at  Strasbourg  have  confirmed  these 
conclusions. 

Bruynoghe  and  Maisin  have  treated  with  success  affections  due  to  the 
staphylococcus  and  anthrax  by  the  subcutaneous  injection  of  cultures  of  the 
bacteriophage. 

Beckerich  and  Hauduroy  have  experimented  with  typhoid  fever  and  with 
pyelocystitis.  They  have  employed  cultures  of  the  bacteriophage  virulent 
for  the  causative  bacterium,  heated  to  58°C.  for  thirty  minutes.  They 
record  the  following  cases; 

1.  An  adult  with  typhoid  fever  of  moderate  severity.     The  patient 
ingested  2  cc.  of  bacteriophage  culture  on  the  18th  day  of  the  disease. 
Defervescence  took  place  forty-eight  hours  later. 

2.  An  adult  with  typhoid  fever  of  moderate  severity.     The  patient 
received  the  same  treatment,  with  the  same  results.    The  culture  was 
given  on  the  ninth  day  of  the  disease. 

3.  An  infant  with  severe  typhoid  fever.    The  patient  was  given  2  cc. 
of  the  bacteriophage  culture  by  mouth  and  in  addition  the  simultaneous 
injection  of  1  cc.    This  treatment  was  given  on  the  twentieth  day.    The 
temperature  came  down  within  forty-eight  hours  and  the  apyrexia  was 
permanent. 

4.  An  infant  with  a  paratyphoid  B  infection,  whose  condition  was  grave. 
On  the  ninth  day  the  bacteriophage  was  given  by  the  simultaneous  inges- 
tion  and  injection  of  the  culture.    After  the  next  day  the  apyrexia  was 
permanent. 

5.  An  infant  with  paratyphoid  B  infection  of  average   severity.    On 
the  23rd  day  the  bacteriophage  was  given  by  ingestion  and  injection. 
The  apyrexia  was  permanent  after  the  next  day. 

Two  adults  affected  with  typhoid  fever  of  the  ataxo-adynamic  form  and 
with  pronounced  myocardial  involvement  were  treated.  The  apyrexia 
which  followed  the  treatment  within  forty-eight  hours  did  not  prevent 
death.  Considering  that  in  these  two  cases  the  failure  might  be  due,  either 
to  a  too  delayed  intervention,  or  to  too  small  a  dose  in  view  of  the  severity 
of  the  cases,  they  decided  to  increase  the  quantity  of  bacteriophage  culture 
administered  by  ingestion  in  such  cases. 

6.  An  infant  affected  with  typhoid  fever  in  very  severe  form.    On  the 
tenth  day  the  patient  was  given  5  cc.  of  culture  by  mouth  and  a  simul- 
taneous injection  of  1  cc.     Within  forty-eight  hours  there  was  permanent 
defervescence  with  euphoria. 

7.  An  infant  with  a  very  severe  case  of  typhoid  fever.     The  same  treat- 
ment was  given  on  the  fourteenth  day.     Defervescence  with  euphoria 
occurred  in  forty-eight  hours. 


IMMUNIZATION   BY  MEANS   OF  THE  BACTEKIOPHAGE  271 

These  authors  confirm  the  absolute  harmlessness  of  the  administration 
of  cultures  of  the  bacteriophage,  whatever  may  be  the  mode  of  introduction 
into  the  organism.  The  sole  reaction  which  they  observed  consisted  of  a 
sudoral  crisis  which  followed  the  administration  in  about  two  hours, 
even  when  the  administration  was  effected  by  the  oral  route.  They  con- 
sider that  this  reaction  is  due  to  the  lysis  of  the  pathogenic  bacteria  within 
the  body  of  the  patient.  This  view  is  certainly  correct  for  this  sudoral 
crisis  is  not  observed  in  the  healthy  individual,  either  after  the  injection 
or  after  the  ingestion  of  cultures  of  the  bacteriophage.  This  has  been 
demonstrated  in  several  instances. 

They  note  the  constant  coincidence  which  exists  between  the  admin- 
istration of  the  culture  and  the  apyrexia  which  follows  within  forty-eight 
hours.  This  relationship  appears  to  be  independent  of  the  stage  of  the 
disease  when  the  culture  is  given.  In  all  the  treated  cases  blood  cultures 
made  forty-eight  hours  before  the  intervention  were  positive,  that  is,  the 
treatment  was  always  applied  at  a  period  when  the  disease  was  fully  active. 

Beckerich  and  Hauduroy  have,  moreover,  treated  two  cases  of  puer- 
peral pyelocystitis  due  to  B.  coli  by  the  subcutaneous  injection  of  1  cc.  of  an 
anti-coli  bacteriophage  culture.  In  both  cases  the  sudoral  crisis  followed 
in  two  hours,  and  permanent  apyrexia  in  forty-eight  hours. 


CHAPTER   IV 

THE  BACTERIOPHAGE  AND  IMMUNITY 
SUMMARY  AND  CONCLUSIONS 

The  bacteriophage,  Bacteriophagum  intestinale  d'Herelle,  1918, 
an  ultramicrobial  parasite  of  bacteria,  normally  exists  in  the 
intestinal  tracts  of  animals,  both  vertebrates  and  invertebrates. 
The  possibility  of  counting  the  ultramicrobes  is  a  most  important 
point  in  the  study  of  the  bacteriophage,  for  it  makes  it  possible 
to  follow  its  development  and  to  recognize  its  mode  of  action 
in  vitro  and  in  vivo.  An  obligatory  parasite,  the  bacteriophage 
lives  only  at  the  expense  of  living,  normal  bacteria,  which  con- 
stitute its  sole  culture  medium.  Experiments  and  ultramicro- 
scopic  examination  agree  in  showing  that  the  ultramicrobial 
bacteriophage  penetrates  into  the  interior  of  the  bacterium,  there 
forms  a  colony  of  fifteen  to  twenty-five  elements  within  one  to 
one  and  one-half  hours;  whereupon  the  bacterium  bursts  and 
liberates  the  young  ultramicrobes.  For  its  development  the 
bacteriophage  utilizes  the  substance  of  the  bacteria  which  it 
dissolves  by  the  aid  of  the  lytic  diastases  which  it  secretes.  The 
property  possessed  by  the  bacteriophage  of  secreting  a  lysin, 
active  for  a  given  bacterium, — that  which  permits  it  to  penetrate 
this  bacterium  and  to  reproduce  there — represents,  in  the  strict 
sense  of  the  word,  its  virulence  for  this  bacterium. 

There  is  but  a  single  species  of  bacteriophage,  common  to  all 
animals,  capable  of  acquiring  virulence  for  different  bacterial 
species,  probably  for  all  species. 

Just  as  for  each  pathogenic  bacterium  there  is  a  scale  of  viru- 
lence for  a  given  animal,  so  also  for  each  bacteriophage  there  is 
an  individual  virulence.  We  are  able  to  increase  or  attenuate 
the  virulence  of  the  pathogenic  bacterium,  and  the  same  phenom- 
enon can  be  obtained  with  the  bacteriophage.  The  parasitized 
superior  organism  defends  itself  against  the  bacterial  secretions 
and  is  able  to  acquire  an  antitoxic  immunity;  the  bacterium  at- 

272 


THE   BACTERIOPHAGE   AND   IMMUNITY  273 

tacked  by  the  bacteriophage  does  not  remain  passive.  It  resists, 
and  is  able  to  overcome  the  ultramicrobe  and  to  acquire  an  anti- 
lytic  immunity.  All  the  vicissitudes  of  the  struggle  between  the 
animal  and  the  bacterium  have  their  counterpart  in  the  struggle 
between  the  parasitizing  bacteriophage  and  the  bacterium  at- 
tacked. The  resemblance  is  complete.  It  is  simply  a  matter  of 
descending  a  degree  in  the  order  of  size  of  the  beings  involved. 

The  existence  of  the  bacteriophage  in  the  intestine  of  all  liv- 
ing beings,  its  exiguity  which  allows  it  to  filter  through  soils 
impermeable  to  bacteria,  its  vitality  and  resistance  to  agents  of 
destruction,  explain  its  extreme  diffusion  in  nature. 

When  derived  from  the  organism  a  single  strain  of  the  bac- 
teriophage is  rarely  active  for  but  a  single  bacterial  species. 
Usually  it  attacks  several  species  at  one  and  the  same  time,  and 
for  each  it  possesses  a  separate  and  variable  virulence.  There  is 
but  one  bacteriophage  but  there  is  an  infinity  of  strains,  each 
possessing,  when  taken  from  the  organism,  the  power  of  attack- 
ing a  certain  number  of  bacteria.  A  single  strain  is  variable  from 
time  to  time,  both  as  to  its  intensity  of  action  against  each  bac- 
terial species,  and  as  to  the  extent  of  its  action  with  regard  to 
the  number  of  bacterial  species  attacked.  All  combinations  of 
virulence  being  possible  in  quantity  as  in  quality,  it  can  be  un- 
derstood in  view  of  the  infinite  number  of  possible  combinations, 
that  there  can  exist  no  two  strains  of  ultramicrobial  bacterio- 
phage which  can  be  exactly  alike. 

In  a  bacterial  suspension  inoculated  with  a  culture  of  an  active 
strain  of  bacteriophage  it  is  not  always  the  latter  which  prevails. 
If  the  bacterium  succeeds  in  acquiring  a  resistance  a  selection  of 
more  and  more  resistant  bacteria  occurs,  resulting  in  the  forma- 
tion of  a  state  of  equilibrium  between  the  resistant  bacterium  and 
the  virulent  bacteriophage  which  then  coexist  in  the  medium. 
A  mixed  culture  results  in  which  the  equilibrium  is  more  or  less 
stable  but  which  can  be  overthrown  in  favor  of  the  one  or  the 
other  of  the  germs  there  present,  according  to  the  circumstances 
of  the  moment. 

The  acquisition  of  resistance  is  accompanied  by  changes  in 
morphology  and  in  the  properties  of  the  bacteria;  the  bacilli 
take  a  coccoid  aspect  and  become  surrounded  by  a  capsule. 


274  THE   BACTEBIOPHAGE 

They  become  inagglutinable.  They  resist  phagocytosis.  They 
are  endowed  with  a  very  great  vitality  and  a  very  high  virulence. 
Loss  in  resistance  is  accompanied  by  a  return  to  normal  form 
and  properties. 

Although  the  bacteriophage  is  capable  of  acquiring  a  virulence 
for  the  bacterium,  the  bacterium  on  its  side  is  capable  of  acquir- 
ing a  resistance  to  the  bacteriophage.  The  virulence  of  the  one 
and  the  resistance  of  the  other  are  not  fixed,  but  are  essentially 
variables,  being  enhanced  or  attenuated  according  to  the  in- 
herited properties  of  each  of  the  two  germs,  and  according  to 
the  circumstances  of  the  moment  which  favor  the  one  or  the  other 
of  the  two  antagonists.  These  two  phenomena  dominate  the 
pathogenesis  and  the  pathology  of  infectious  diseases. 

The  bacteriophagous  ultramicrobe  is  a  normal  inhabitant  of 
the  intestine,  an  obligatory  parasite,  and  there  maintains  itself 
at  the  expense  of  saprophytic  bacteria  hereditarily  endowed  with 
a  certain  resistance,  with  which  it  lives  in  commensalism.  For 
any  bacterium  whatever,  pathogenic  or  not  when  introduced 
into  the  intestine,  the  bacteriophage  exalts  its  virulence  toward 
the  invader,  and  this  so  much  the  more  rapidly  when  this  bac- 
terium is  lacking  in  resistance  and  when  the  bacteriophage  is 
hereditarily  the  more  adapted  to  the  struggle.  The  more  fre- 
quent the  reinfections  by  a  given  bacterium,  the  more  likely  is 
the  bacteriophage  to  quickly  acquire  a  degree  of  virulence  suffi- 
cient to  inhibit  all  growth.  If  the  bacterium  which  invades 
an  individual  is  the  agent  of  an  intestinal  infection  or  if  the 
avenue  of  infection  is  intestinal,  the  infectious  process  is  then 
prevented  at  its  very  inception  and  the  disease  aborts  before 
morbid  symptoms  appear. 

The  rapid  adaptation  of  the  bacteriophage  may  be  delayed  or 
even  prevented  by  unfavorable  circumstances.  More  sensitive 
than  the  bacteria  to  the  action  of  acids  and  alkalies,  the  reaction 
of  the  medium  is  a  principal  factor  which  influences  the  develop- 
ment of  the  bacteriophage  and  its  power  of  attack.  On  the  other 
hand,  its  activity  may  be  annihilated  if  the  invading  germ  is  de- 
rived from  an  organism  in  which  it  has  been  in  conflict  with  the 
bacteriophage,  a  conflict  which  has  allowed  it  to  acquire  some 
degree  of  resistance.  In  either  the  one  or  the  other  of  these  cases 


THE   BACTERIOPHAGE   AND   IMMUNITY  275 

the  pathogenic  bacterium  grows  and  disease  results.  If  the  en- 
vironmental conditions  remain  unfavorable  and  inhibit  the 
action  of  the  bacteriophage,  the  bacterium  develops  freely  and 
the  invaded  individual  succumbs  quickly,  or  the  conflict  may 
become  established,  with  the  virulence  of  the  bacteriophage 
gradually  increasing  by  selection  and  the  resistance  of  the  bac- 
terium likewise  increasing  as  the  result  of  a  similar  selective 
process.  The  condition  of  the  individual  in  which  this  con- 
flict is  taking  place  faithfully  reflects  the  changes  in  the 
struggle.  Convalescence  is  established  only  at  the  moment 
when  the  virulence  of  the  bacteriophage  effectively  dominates 
the  resistance  of  the  bacterium.  If  the  opposite  results,  if  the 
bacterium  acquires  a  refractory  state,  no  further  barrier  is  opposed 
to  the  invasion  of  the  individual  and  death  follows. 

In  a  word,  recovery  is  always  a  result  of  the  exaltation  of  the 
virulence  of  the  bacteriophage,  an  increase  sufficient  to  permit 
it  to  parasitize  and  destroy  the  pathogenic  bacteria  implanted 
in  the  body.  Death  takes  place,  either  as  a  result  of  inertia  in 
the  bacteriophage,  or  because  of  the  acquisition  of  a  refractory 
state  by  the  bacteria,  conditions  which,  in  either  case,  allow  the 
latter  to  develop  without  hindrance. 

There  is,  however,  a  third  possibility.  One  may  isolate  from 
the  intestinal  contents  of  " bacillus  carriers,"  typhoid  or  dysen- 
tery, and  indeed  constantly,  a  resistant  pathogenic  bacterium  and 
a  bacteriophage  virulent  for  this  bacterium.  There  has  been 
a  commensality  (as  is  the  case  with  the  normal  saprophytes  of 
the  intestine),  a  mixed  culture.  Usually  this  equilibrium  is 
quickty  broken  in  favor  of  the  bacteriophage  and  the  carrier  state 
ends.  But  in  individuals  who  become  carriers  the  conflict  car- 
ried on  in  the  intestine  between  the  bacteriophage  and  the  bac- 
terium is  sufficiently  long  to  permit  the  development  of  an  organic 
immunity.  The  bacteria  act  on  the  organism  only  by  means 
of  their  toxins.  A  bacterium  whose  toxin  does  not  exercise 
any  action  on  the  cells  of  an  animal  is  as  inoffensive  for  it  as  a 
bacterium  naturally  atoxic.  From  the  time  when  an  organic 
immunity  is  acquired  by  the  carrier  the  pathogenic  bacterium 
becomes  for  him  a  saprophyte. 

One  can  comprehend,  on  the  other  hand,  the  danger  of  con- 
tamination from  carriers,  who,  although  they  distribute  a  viru- 


276  THE   BACTERIOPHAGE 

lent  bacteriophage  also  at  the  same  time  distribute  a  resistant 
bacterium,  that  is  to  say,  a  bacterium  particularly  apt  to  nega- 
tive the  defense  exercised  by  the  intestinal  bacteriophage  of  the 
susceptible  individuals  contaminated  by  the  carrier. 

The  observations  made  in  pyelonephritis  lead  us  to  believe 
that  the  individual  affected  with  a  chronic  infectious  disease  is 
in  reality  an  internal  carrier.  Here  also,  the  individual  enjoys 
an  antitoxic  immunity.  Nevertheless  there  is  a  struggle  within 
the  organism  between  the  virulent  bacteriophage  and  the  re- 
sistant bacterium.  For  while  in  the  intestine  the  bacterium  is 
henceforth  inoffensive  and  offers  no  great  inconvenience  to  the 
host,  the  presence  of  a  bacterial  culture  within  the  tissues  is  not 
an  indifferent  matter  because  of  the  inflammatory  reactions  which 
it  provokes.  In  addition,  phagocytosis  is  not  able  to  play  an 
active  role,  for  we  have  seen  that  bacteria  which  have  acquired 
a  resistance  to  the  bacteriophage  are  likewise  resistant  to  the 
phagocytic  phenomenon. 

The  r61e  of  the  bacteriophage  is  not  confined  to  the  intestine. 
Whatever  may  be  the  infectious  disease  under  consideration, 
there  is  always  the  introduction  of  the  pathogenic  bacterium  into 
the  intestine,  either  by  the  digestive  path  or  by  the  hepatic  route. 
Thus,  the  intestinal  bacteriophage  may  come  in  contact  with, 
and  acquire  a  virulence  for,  the  pathogenic  bacterium.  Further- 
more, experiment  shows  that  the  bacteriophage  may  enter  the 
circulation  in  the  case  of  a  septicemia.  Hence  it  may  exert  its 
action  at  any  point  in  the  body. 

The  action  of  the  bacteriophage  manifests  itself  in  still  another 
way.  Growing  at  the  expense  of  the  bacteria  it  dissolves  them. 
The  bacterial  substance,  dissolved  and  modified  under  the  action 
of  the  lysins  secreted  by  the  bacteriophage,  is  in  a  physical  and 
chemical  state  particularly  suited  to  act  upon  the  cells  of  the 
organism  which  elaborate  the  antitoxins. 

Finally,  experiment  shows  that  the  bacteriophage  exercises 
a  preponderant  action  on  phagocytosis.  On  one  side  is  the 
fact  that  the  bacteriophage  through  its  lysins  possesses  an  ex- 
tremely high  opsonic  power1  and  on  the  other  side,  a  bacterium 

1  May  not  the  lysins  of  the  bacteriophage  and  the  antilysins  of  the  bac- 
teria be  synonyms  for  opsonins  and  aggressins? 


THE  BACTERIOPHAGE  AND  IMMUNITY  277 

resisting  the  bacteriophage  is,  by  this  fact,  also  resistant  to  phago- 
cytosis. 

The  bacteriophage,  the  direct  agent  of  antimicrobial  immunity 
which  is  by  its  nature  heterologous,  at  least  in  the  sensitive 
animal,  is  also  indirectly  an  agent  of  organic  immunity,  by  na- 
ture homologous. 

The  history  of  the  disease  is  in  effect  the  history  of  the  con- 
flict between  the  bacteriophage  and  the  bacterium.  We  can 
observe  that  the  same  facts  hold  true,  but  on  a  larger  scale, 
in  the  history  of  an  epidemic.  The  virulent  ultramicrobe, 
which  is  present  in  the  intestine  of  all  convalescents,  is  dis- 
seminated by  them  with  their  dejections.  It  is  then  capable 
of  "contaminating"  susceptible  neighboring  individuals  quite 
regardless  of  whether  the  disease  with  which  it  is  associated  is 
intestinal,  septicemic,  or  localized  in  its  nature. 

Observation  shows  that  in  the  last  analysis  the  history  of  an 
epidemic  registers  the  variations  in  the  struggle  between  the 
two  agents,  the  pathogenic  bacterium  and  the  bacteriophagous 
ultramicrobe.  It  is  also  clear  that  the  latter  is  transmissible 
from  individual  to  individual.  The  immunity  is  contagious  in 
the  same  degree  as  is  the  disease  itself.  The  beginning  of  an 
epidemic  is  marked  by  the  diffusion  of  a  bacterium  whose  viru- 
lence is  increased  progressively  by  passages  through  susceptible 
individuals.  Thus  the  epidemic  extends.  In  its  turn  the  ul- 
tramicrobial  bacteriophage  increases  in  virulence  for  the  patho- 
genic bacterium,  and  extends  equally.  The  epidemic  ceases 
when  all  susceptible  individuals  have  been  infected  by  the  viru- 
lent bacteriophage. 

We  have  seen  that  the  bacteriophage  may  conserve  for  a  long 
time  a  "latent"  virulence  for  a  given  bacterium.  These  latent 
virulences,  maintained  moreover  by  accidental  contaminations, 
explain  the  difference  which  exists  between  the  mode  of  propaga- 
tion of  sporadic  diseases  and  of  epidemic  diseases.  Against  the 
bacteria,  agents  of  the  first,  the  bacteriophage  is  always  ready 
to  intervene,  and  it  is  only  exceptionally  that  infection  is  followed 
by  disease.  In  the  second,  on  the  contrary,  particularly  since 
the  agent  is  most  often  imported,  the  bacteriophage  does  not  at 
the  beginning  possess  a  specific  virulence.  The  epidemic  extends 


278  THE   BACTEKIOPHAGE 

and  only  stops,  or  assumes  a  sporadic  character,  when  there  has 
been  a  diffusion  of  a  bacteriophage  virulent  for  the  pathogenic 
bacterium. 

The  bacteriophagous  ultramicrobe  virulent  for  a  given  bac- 
terium is  cultivable  in  vitro.  It  is  therefore  possible  to  obtain 
it  in  any  desired  amount.  If  its  protective  power  is  real  a  sus- 
ceptible individual  should  be  rendered  immune  by  inoculation, 
just  as  though  he  had  naturally  resisted  the  contagion.  This 
has  been  demonstrated  to  be  the  case  in  the  experiments  made 
on  avian  typhosis  and  in  hemorrhagic  septicemia  in  the  buffalo. 

The  injection  of  an  individual  with  a  culture  of  the  bacterio- 
phage virulent  for  a  given  bacterium  is  harmless  and  causes  no 
reaction,  even  when  the  bacteriophage  has  developed  at  the  ex- 
pense of  a  highly  toxic  bacterium — B.  dysenteriae  or  B.  pestis, 
for  example.  The  injected  ultramicrobes  pass  quickly  into 
the  intestine. 

The  injection  of  a  culture  of  the  bacteriophage  provokes  two 
types  of  immunity,  heterologous  and  homologous. 

The  heterologous  antimicrobial  immunity  is  effective  immediately. 
Indeed,  it  exists  simply  by  virtue  of  the  presence  in  the  body  of  a 
bacteriophage  active  for  the  causative  bacterium.  In  an  un- 
contaminated  or  non-epidemic  area  this  immunity  is  transitory. 
In  a  contaminated  or  epidemic  area  it  persists  as  long  as  rein- 
fections occur. 

The  homologous,  or  organic  immunity,  develops  after  an  incu- 
bation period.  It  results  from  the  production  of  specific  anti- 
bodies, most  likely  substances  similar  to  the  antitoxins.  These 
antibodies  are  detectable  in  the  serum  of  the  immunized  animal 
and  persist  there  for  a  period  at  present  undetermined  (more 
than  seven  months  in  the  case  of  avian  typhosis).  The  period 
of  incubation  in  the  homologous  immunity  is  the  longer  as  the 
dose  injected  is  greater. 

The  immunization  experiments  against  barbone  have  shown 
us  the  importance  of  the  question  of  dosage.  The  quantity  of 
bacteriophage  culture  necessary  and  sufficient  to  provoke  or- 
ganic immunity  ought,  in  all  cases,  to  be  injected  at  a  single  time. 
As  to  the  dose  itself,  it  must  certainly  vary  in  accordance  with 
the  disease  under  consideration,  and,  consequently,  with  the 


THE  BACTERIOPHAGE  AND   IMMUNITY  279 

bacterium  for  which  the  bacteriophage  injected  is  virulent.2 
In  hemorrhagic  septicemia  of  the  buffalo  the  optimum  single 
dose  is  a  quarter  of  a  cubic  centimeter  per  hundred  kilograms  of 
body  weight.  This  question  of  dosage  must  be  fixed  by  prelimi- 
nary experiments  for  the  other  diseases.3 

With  disease  once  declared,  the  introduction  into  the  patient 
of  the  ultramicrobe  virulent  for  the  causative  bacterium  ought 
to  place  the  affected  individual  in  a  condition  analogous  to  that 
of  the  convalescent  individual.  The  experiments  in  avian  ty- 
phosis  and  in  human  dysentery  show  in  effect  that  the  ingestion 
or  the  injection  of  cultures  of  the  bacteriophage  exert  a  curative 
action. 

The  administration  to  a  patient  of  an  active  culture  of  bac- 
teriophage ought,  as  may  be  conceived,  to  be  made  at  a  time  as 
near  as  possible  to  the  beginning  of  the  disease.  For  this  there 
are  two  reasons. 

1.  We  have  seen  that  the  acquisition  of  virulence  in  the  bac- 
teriophage only  represents  one  side  of  the  question  of  recovery. 
The  bacterium  may  acquire  a  state  of  resistance  such  that  the 
action  of  the  bacteriophage  may  be  rendered  inoperative.     The 
administration  of  a  culture  of  the  active  bacteriophage  should 
have  the  more  effect  when  the  resistance  of  the  bacterium  is  the 
least.     On  the  other  hand,  the  acquisition  of  resistance  is  the 
result  of  the  conflict  within  the  individual.     Thus,  the  more 
rapid  the  intervention  the  less  likely  will  be  the  formation  of 
a  resistant  bacterial  race. 

2.  If  there  exists  at  the  time  of  intervention  organic  lesions 
incompatible  with  life  the  issue  of  the  disease  can  not  be  other 
than  fatal  whatever  the  power  of  the  bacteriophage.4 

2  It  should  be  noted  that  here  we  are  dealing  with  injection  only.     The 
ingestion  of  cultures  of  the  bacteriophage  does  not  appear  to  be  attended 
by  the  development  of  an  organic  immunity.     Ingestions  can  be  repeated 
without  inconvenience,  as  I  have  demonstrated  on  myself. 

3  It  should  be  emphasized  that  the  cultures  of  the  bacteriophage  used  in 
immunization  should  be  perfectly  limpid;  that  is  to  say,  the  lysis  ought 
to  be  complete.    Filtration  through  a  bougie  is  essential,  for  the  reason 
which  we  have  seen.     If  necessary,  filtration  may  be  replaced  by  heating 
at  58°C.,  but  filtration  is  to  be  preferred. 

4  The   cases  recently  described  by  Beckerich  and  Hauduroy,  which 
were  mentioned  in  the  note  at  the  end  of  the  preceding  chapter,  corroborate 


280  THE   BACTEBIOPHAGE 

To  summarize:  Observation  and  experiment  agree  in  showing 
that  the  bacteriophage  is  the  direct  agent  of  antibacterial  immunity 
in  the  sensitive  animal.  It  dissolves  the  bacteria  at  the  expense 
of  which  it  reproduces  itself,  and  does  this  by  means  of  lysins 
which  it  secretes  and  which  remain  in  the  solution  once  the  bac- 
teria are  destroyed.  These  lysins  enjoy,  furthermore,  an  extremely 
high  opsonic  power,  which  may  likewise  contribute,  in  certain 
cases,  to  the  destruction  of  the  pathogenic  bacteria. 

The  bacteriophage  also  contributes  to  the  establishment  of 
organic  immunity.  The  bacterial  substance  dissolved  under 
the  action  of  the  lysins  is  in  a  physical  and  chemical  state  such 
that  an  extremely  minute  quantity  suffices  to  provoke  the  forma- 
tion of  a  potent  organic  immunity. 

this  statement.  I  state  precisely  then,  and  I  insist  on  this  point,  that 
the  treatment  by  the  bacteriophage  of  any  case  of  acute  infection  ought 
to  be  undertaken  at  once,  without  the  loss  of  a  minute.  Whatever  may  be 
the  disease  it  is  useless  and  dangerous  to  await  the  results  of  laboratory 
examinations  destined  to  confirm  the  clinical  diagnosis.  This  last  ought 
to  be  considered  as  sufficient  to  warrant  the  administration  of  the  bacterio- 
phage. Such  practice  does  not  incur  any  risk  whatever,  even  though  there 
has  been  an  error  in  the  diagnosis,  for  the  injection  or  the  ingestion  of 
cultures  of  the  bacteriophage  is  in  all  cases  absolutely  innocuous.  I  would 
say  further,  even  if  the  clinical  diagnosis  proves  erroneous  the  adminis- 
tration of  a  bacteriophage  avirulent  for  the  causative  bacterium  may  be 
useful.  While  in  Indo-China,  at  three  different  times,  I  administered  to 
cholera  patients,  per  os,  two  cubic  centimeters  of  an  anti-Shiga  bacterio- 
phage, and  two  of  the  three  cases  recovered.  I  do  not  affirm  that  this 
fortunate  result  could  be  referred  to  the  administration  of  the  anti-Shiga 
bacteriophage,  although  there  is  a  strong  presumption  in  favor  of  this 
hypothesis.  In  fact,  of  the  113  cases  of  cholera  which  I  observed  during 
my  stay  there,  I  did  not  see  a  single  case  recover  spontaneously.  In  any 
event,  even  if  it  be  but  a  coincidence,  it  is  possible  to  affirm  that  the  admin- 
istration of  the  bacteriophage  caused  no  harm  in  the  cholera  cases. 

In  so  far  as  typhoid  fever  is  concerned,  for  example,  I  would  recommend 
the  removal  of  the  blood  necessary  for  culture  at  the  entrance  of  the  patient 
into  the  hospital  (or  at  the  first  visit  of  the  physician  treating  the  case) 
and  the  immediate  administration  of  a  culture  of  bacteriophage.  Either 
two  or  five  cubic  centimeters  may  be  given  per  os,  or  one  cubic  centimeter 
may  be  injected  subcutaneously.  In  this  way  a  bacteriologic  diagnosis 
may  be  established  without  necessitating  delay  in  treatment.  In  a  word, 
whatever  may  be  the  disease,  the  absolute  principle  ought  to  be  "never 
lose  a  minute." 


THE   BACTERIOPHAGE  AND  IMMUNITY  281 

The  immunity  acquired  as  the  result  of  a  single  injection  of  a 
small  quantity  of  bacteriophage  culture  is  accompanied  by  the 
appearance  in  the  blood  of  a  protective  principle.  The  animal 
which  receives  this  blood  enjoys  a  solid  immunity,  specific  in 
nature,  and  identical  with  that  possessed  by  the  animal  which 
received  the  immunizing  injection  of  bacteriophage.  The  pro- 
tective principle  is  probably  an  antitoxin.  It  is  possible  that 
this  new  method  of  obtaining  immunizing  sera  offers  a  means  of 
intervening  in  the  course  of  a  disease,  even  in  cases  where  the 
administration  of  the  active  culture  of  bacteriophage  may  be 
without  effect  because  of  the  previous  acquisition  of  a  resistance 
by  the  bacterium. 

Our  knowledge  of  the  bacteriophage,  a  cultivable  agent  of 
immunity,  allows  us  to  entertain  the  possibility  of  collective  in- 
tervention in  epidemics. 

Whatever  may  be  the  epidemic  (provided,  of  course,  the  agent 
is  known  and  cultivable)  we  have  first  the  possibility  of  individual 
vaccination  by  means  of  a  single  injection  of  a  small  quantity  of 
bacteriophage  culture  active  for  the  causative  bacterium.  But 
we  have  seen  that  the  presence  in  the  intestine  of  active  ultrami- 
crobes  assures  the  protection  of  a  susceptible  individual.  We 
are  then  able  to  consider  the  possibility  of  collective  immuniza- 
tion of  the  population,  for  it  would  be  easy  to  mix  cultures  of 
the  bacteriophage  with  the  drinking  water,  especially  in  urban 
centers.  One  might  then  be  assured  of  an  active  bacteriophage 
in  the  intestine  of  all  susceptible  individuals  throughout  the 
critical  period.  The  method  offers  no  risk;  the  cultures  can  be 
ingested  without  inconvenience  in  any  quantity. 

In  spite  of  the  fact  that  I  have  specified  at  several  times  in 
the  course  of  this  work  that  the  experiments  undertaken  deal 
only  with  antibacterial  immunity  in  the  susceptible  individual, 
I  want  in  closing,  in  order  to  avoid  confusion,  to  say  a  few  words 
on  the  subject  of  phagocytosis,  for  it  would  be  strange  if,  speaking 
of  antibacterial  immunity,  I  made  no  allusion  to  this  mode  of 
defense. 

I  do  not  oppose  any  of  the  conclusions  of  Metchnikoff  touching 
the  role  of  phagocytosis  in  the  natural  immunity  which  character- 
izes the  refractory  state.  In  acquired  immunity  also,  Metchni- 


282  THE  BACTERIOPHAGE 

koff  and  his  collaborators  affirm  that  phagocytosis  plays  a 
capital  r61e.  That  the  elimination  of  bacteria  is  effected  by 
phagocytosis,  once  organic  immunity  is  established,  would  appear 
to  be  the  proper  interpretation. 

However,  in  the  one  or  in  the  other  case,  the  bacteriophage 
manifests  its  action.  Its  activity  is  not  naturally  limited  to  the 
bacteria  pathogenic  for  a  given  animal;  it  is  exercised  without 
distinction  against  bacteria  pathogenic  and  saprophytic,  in  all 
circumstances,  and  in  all  animals.  Even  if,  in  the  immune 
animal,  the  bacteriophage  should  remain  inert,  the  bacteria  would 
none  the  less  be  eliminated  by  phagocytosis. 

What  then  is  the  role  of  the  bacteriophage  in  immunity?  The 
defense  of  the  susceptible  individual  exposed  to  infection,  and  the 
protection  of  the  organism  in  the  course  of  natural  disease.  Par- 
asitic of  bacteria,  the  bacteriophage  intervenes  directly  to  destroy 
the  pathogenic  bacteria  which  venture  to  invade  the  organism; 
secreting  lysins  endowed  with  a  powerful  opsonic  action,  it  ren- 
ders possible  the  education  of  the  phagocyte  and  introduces  the 
establishment  of  organic  antibacterial  immunity;  dissolving  the 
bacteria,  it  transforms  the  bacterial  substance  and  places  it  in  a 
physical  and  chemical  state  where  it  can  stimulate  the  cells  of 
the  body  which  produce  the  antitoxic  antibodies,  it  introduces 
thus,  the  establishment  of  organic  antitoxic  immunity. 

In  other  terms,  the  bacteriophage  plays  a  preponderating  role 
in  all  the  phenomena  of  immunity  which  are  accomplished  in  a 
susceptible  individual.  As  a  result  of  its  presence  it  follows  that, 
although  exposed  to  infection  it  is  possible  to  remain  unharmed; 
and  although  sick,  it  is  possible  to  recover. 


BIBLIOGRAPHY 

LIST    OF    COMMUNICATIONS  ON  THE   BACTEBIOPHAGE    BY 
F.  D'  HERELLE  AND  His  ASSOCIATES 

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(10)  D'HERELLE,  F. :  Sur  le  microbe  bacteriophage.    Compt.  rend.  Soc.  de 

biol.,  1920,83,247. 

(11)  D'HERELLE,  F. :  Sur  le  microbe  bacteriophage.    Compt.  rend.  Soc.  de 

biol.,  1920,  83,  1318. 

(12)  D'HERELLE,   F. :  Sur  la  nature  du  principe  bacteriophage.    Compt. 

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(13)  BABLET,  J. :  Sur  le  principe  bacteriophage  de  d'Herelle.    Compt.  rend. 

Soc.  de  biol.,  1920,83,1322. 

(14)  D'HERELLE,  F. :  Le  microbe  bacteriophage,  agent  d'immunite  dans  la 

pest  et  le  barbone.     Compt.  rend.  Acad.  sci.,  1921,  172,  99. 

(15)  D'HERELLE,    F. :  Sur    la   nature   du   bacteriophage    (Bacteriophagum 

intestinale  de  d'Herelle,  1918).    Compt.  rend.  Soc.  de  biol.,  1921, 
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(16)  D'HERELLE,  F. :  Phenomenes  coincidant  avec  V 'acquisition  de  la  resist- 

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de  biol.,  1921,  84,  384. 

283 


284  THE   BACTERIOPHAGE 

(17)  D'HERELLE,  F. :  R6le  du  bacteriophage  dans  I'immunite.    Compt.  rend. 

Soc.  de  biol.,  1921,  84,  538. 

(18)  D'HERELLE,   F.   AND  ELIAVA,   G. :  Sur  le  serum  anti-bacteriophage. 

Compt.  rend.  Soc.  de  biol.,  1921,  84,  719. 

(19)  ELIAVA,  G.  AND  POZERSKI,  E.:  Sur  les  caracteres  nouveaux  presentes 

par  le  bacille  de  Shiga  ayant  resiste  a  I'action  du  bacteriophage  de 
d'Herelle.  Compt.  rend.  Soc.  de  biol.,  1921,  84,  708. 

(20)  D'HERELLE,    F. :  Sur   I'historique   du   bacteriophage.    Compt.    rend. 

Soc.  de  biol.,  1921,  84,  863. 

(21)  D'HERELLE,  F. :  Sur  la  nature  du  bacteriophage.    Compt.  rend.  Soc.  de 

biol.,  1921,  84,  908. 

(22)  D'HERELLE,  F. :  Le  bacteriophage:  son  rdle  dans  I'immunite.    Presse 

meU,  1921,  29,463. 

(23)  ELIAVA,   G.   AND  POZERSKI,  E.:  De  V action  destructive  des  sels  de 

quinine  sur  le  bacteriophage  de  d'Herelle.  Compt.  rend.  Soc. 
de  biol.,  1921,  86,  139. 

(24)  D'HERELLE,  F.:  Le  bacteriophage.    La  Nature,  1921,  Oct.  1,  p.  219. 

(25)  D'HERELLE,   F.   AND  ELIAVA,    G. :  Unicite  de  bacteriophage:  sur   la 

lysine  du  bacteriophage.     Compt.  rend.  Soc.  de  biol.,  1921,  85, 701. 

(26)  D'HERELLE,  F. :  L'ultramicrobe  bacteriophage.    Compt.  rend.  Soc.  de 

biol.,  1921,85,767. 

(27)  D'HERELLE,  F.  AND  POZERSKI,  E.:  Action  de  la  temperature  sur  le 

bacteriophage.    Compt.  rend.  Soc.  de  biol.,  1921,  85,  1011. 

(28)  D'HERELLE,   F. :  Sur  les   anti-lysins   d'origine  bacterienne.    Compt. 

rend.  Soc.  de  biol.,  1922,  86,  360. 

(29)  D'HERELLE,  F. :  Sur  la  presence  du  bacteriophage  dans  les  leucocytes. 

Compt.  rend.  Soc.  de  biol.,  1922,  86,  477. 

COMMUNICATIONS  BY  OTHER  AUTHORS 

(30)  DAMADE,  E.:  Le  microbe  filtrant  bacteriophage  de  d'Herelle.    These 

(in  medicine),  Bordeaux,  1919. 

(31)  KABESHIMA,  T. :  Recherches  experimental  sur  la  vaccination  preventive 

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